Gray leaf spot tolerant maize and methods of production

ABSTRACT

The invention relates to methods and compositions for identifying maize plants that have newly conferred tolerance or enhanced tolerance to, or are susceptible to, Gray Leaf Spot (GLS). The methods use molecular genetic markers to identify, select and/or construct tolerant plants or identify and counter-select susceptible plants. Maize plants that display newly conferred tolerance or enhanced tolerance to GLS that are generated by the methods of the invention are also a feature of the invention.

This application is a continuation of application Ser. No. 13/729,196,filed Dec. 28, 2012 and now U.S. Pat. No. 8,841,509, which is adivisional application of application Ser. No. 12/336,624, filed Dec.17, 2008 and now granted as U.S. Pat. No. 8,367,899, which claims thebenefit of U.S. Provisional Application No. 61/009,697, filed Dec. 31,2007, the disclosure of which is hereby incorporated in its entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named20140917_BB1691USCNT_SequenceListing created on Sep. 17, 2014 and havinga size of 35 kilobytes and is filed concurrently with the specification.The sequence listing contained in this ASCII formatted document is partof the specification and is herein incorporated by reference in itsentirety.

FIELD OF THE INVENTION

This invention relates to Gray Leaf Spot (GLS) tolerant maize plants anda method of producing same. More particularly this invention relates toidentifiable genetic material capable of causing tolerance to GLS inmaize, and the introgression of this genetic material into maize plants.Additionally, the present invention relates to the introgression ofdesired genetic material from one or more parent plants into progenyplants with speed, precision, and accuracy.

BACKGROUND OF THE INVENTION

Historically, maize (corn) is an important crop for food, feed, andindustrial uses. Any environmental stress factor, e.g. disease, thataffects maize can have an impact on maize grain availability for theseuses.

Gray Leaf Spot (hereinafter referred to as GLS) has gained prominencethe last three decades and is a significant foliar disease in the UnitedStates and in other major corn producing areas, such as Mexico, Brazil,Europe, and South Africa. The incidence and severity of GLS appears tobe increasing in the United States (Wang et al., Phytopathology88:1269-75 (1998)), perhaps due to an increase of maize on maizeplantings and reduced tillage. These conditions can contribute tooverwintering of the fungus and early infection the following season(Laterall and Rossi, Plant Dis. 67:842-37 (1983)). Yield losses inexcess of 50% have been reported during GLS epidemics in the UnitedStates (Laterall and Rossi, supra; Lipps, Plant Dis. 71:281 (1987)), andestimated losses have been as high as 100% where severe epidemicscontributed to increased stalk lodging and early senescence (Lateralland Rossi, supra).

The fungal pathogen Cercospora zeae-maydis, which causes GLS,characteristically produces long, rectangular, grayish-tan leaf lesionswhich run parallel to the leaf veins (Tehon and Daniels, Mycologia17:240-49 (1925); Latterell and Rossi, supra; Ward et al., Plant Dis.83:884-95 (1999)). The lesions may blight part or all of the leaf andtypically appear in the lower leaves first. Blighting due to GLS isassociated with the premature loss of photosynthetic area. The dominantsink of the post-flowering maize plant is the ear, and blighting inducesthe plant to transfer photosynthate from the stalk and roots to the ear,at high levels, thus causing premature senescence and reduced yield.

The fast and effective development of maize varieties with GLS toleranceis beneficial. The level of tolerance to GLS in commercial hybrids andinbreds differs among varieties. Some varieties exhibiting strongtolerance have been reported. However, the use of phenotypic selectionto introgress the GLS trait from a tolerant variety into a susceptiblevariety can be time consuming and difficult. GLS is sensitive toenvironmental conditions and requires high humidity and extended leafwetness. This sensitivity makes it difficult to reliably select for GLStolerance from year to year based solely on phenotype (Lehmensiek etal., Theor. Appl. Genet. 103:797-803 (2001)). Specialized diseasescreening sites can be costly to operate, and plants must be grown tomaturity in order to classify the level of tolerance. In contrast,selection through the use of molecular markers associated with GLStolerance has the advantage of permitting at least some selection basedsolely on the genetic composition of the progeny. Thus, GLS tolerancecan be measured very early on in the plant life cycle, even as early asthe seed stage. The increased rate of selection that can be obtainedthrough the use of molecular markers associated with the GLS tolerancetrait means that plant breeding for GLS tolerance can occur at a fasterrate and that commercially acceptable GLS tolerant plants can bedeveloped more quickly.

SUMMARY OF THE INVENTION

Embodiments of this invention are based on the fine mapping of geneticloci significantly correlated with increased GLS tolerance, and theapplication of this knowledge to plant breeding. Compositions andmethods for identifying maize plants with tolerance to GLS are provided.Methods of making maize plants that are tolerant to GLS through markerassisted breeding are provided, as well as plants produced by suchmethods.

Embodiments include an improved donor variety PHJEP for use as a sourceof germplasm to introgress tolerance to GLS into maize plants, andprogeny derived therefrom. A representative sample of said variety hasbeen deposited with American Type Culture Collection (ATCC) as AccessionNumber PTA-8851.

One aspect is for a seed of a maize variety designated PHJEP, wherein arepresentative sample of said maize variety has been deposited as ATCCaccession number PTA-8851, or a progeny seed derived therefrom thatcomprises the PHJEP gray leaf spot tolerance locus and that, when grown,produces a plant that exhibits gray leaf spot tolerance. Plants producedfrom PHJEP seed or the seed of its progeny are also of interest, as arecells of those plants.

Embodiments also include the specific recombinant chromosomal intervalobtained in PHJEP correlated with enhanced GLS tolerance, and theintrogression of this chromosomal interval into other varieties andplants. Some embodiments include the introgression of unique haplotypesof PHJEP into other varieties and plants.

In one aspect, the PHJEP gray leaf spot tolerance locus in the progenyseed is located on a PHJEP-derived chromosomal interval comprising achromosomal region of PHJEP defined by UMC1346 and UMC1702. In another,the PHJEP gray leaf spot tolerance locus in the progeny seed is definedby a haplotype comprising: allele G at PHM 00045-01, allele A at PHM00043-01, allele A at PHM 15534-13, allele G at PHM 04694-10, allele TatPHM 01811-32, allele Tat PHM 01963-15, allele C at PHM 01963-22, alleleTat PHM 05013-12, allele Tat PHM 00586-10, allele A at PHM 00049-01. Instill another aspect, the PHJEP gray leaf spot tolerance locus isdefined by a haplotype comprising: allele G at PHM 00045-01, allele A atPHM 00043-01, allele C at PHM 01963-22, and allele T at PHM 05013-12.

In other aspects, the progeny seed is a backcross conversion of thePHJEP gray leaf spot tolerance locus. Also of interest is a progeny seedthat is a backcross conversion produced with a recurrent parent selectedfrom PHVNV, PHW3Y, PHVRA, PHEWB, and PHWRC.

In other aspects the progeny seed is a hybrid variety, and at least oneinbred parent of the hybrid variety is a backcross conversion of thePHJEP gray leaf spot tolerance locus into a recurrent parent selectedfrom PHVNV, PHW3Y, PHVRA, PHEWB, and PHWRC.

Other embodiments include a process for identifying a first corn plantcomprising a locus correlated with gray leaf spot tolerance, saidprocess comprising: (a) obtaining a first genetic profile of said firstcorn plant for the chromosomal interval on chromosome 4 between BNLG1755and UMC1299, (b) obtaining a second genetic profile from a second cornplant comprising the locus correlated with gray leaf spot tolerance,wherein the locus is located on chromosome 4 between BNLG1755 andUMC1299, and (c) comparing said first genetic profile with said secondgenetic profile.

In another aspect, the process further comprises selecting said firstcorn plant if it comprises the locus correlated with gray leaf spottolerance.

In addition, the second genetic profile can be the genetic profile ofPHJEP or a progeny of PHJEP, or can comprise one or more marker allelesselected from the group consisting of: allele G at PHM 00045-01, alleleA at PHM 00043-01, allele A at PHM 15534-13, allele G at PHM 04694-10,allele Tat PHM 01811-32, allele Tat PHM 01963-15, allele Cat PHM01963-22, allele Tat PHM 05013-12, allele Tat PHM 00586-10, and allele Aat PHM 00049-01.

The second genetic profile can also comprise one or more marker allelesselected from the group consisting of: allele G at PHM 00045-01, alleleA at PHM 00043-01, allele C at PHM 01963-22, and allele T at PHM05013-12.

In other aspects, the genetic profiles can be determined for thechromosomal interval on chromosome 4 between BNLG1755 and MMC0371. Inaddition, the locus correlated with gray leaf spot tolerance can be PHM15534, PHM 04694, PHM 01811, PHM 01963, PHM 05013, or PHM 00586.

Also of interest are corn plants identified by the process and cells andseeds of those corn plants.

A further embodiment includes a process for identifying a first cornplant comprising a locus correlated with gray leaf spot tolerance, saidprocess comprising: (a) obtaining a first genetic profile of said firstcorn plant for the chromosomal interval on chromosome 4 delineated byand including SEQ ID NO:86, or a nucleotide sequence that is 95%identical to SEQ ID NO:86 based on the Clustal V method of alignment,and SEQ ID NO:87, or a nucleotide sequence that is 95% identical to SEQID NO:87 based on the Clustal V method of alignment, (b) obtaining asecond genetic profile from a second corn plant comprising the locuscorrelated with gray leaf spot tolerance, wherein the locus is in saidinterval, and (c) comparing said first genetic profile with said secondgenetic profile.

Other embodiments include a corn seed comprising a haplotype of: alleleG at PHM 00045-01, allele A at PHM 00043-01, allele A at PHM 15534-13,allele G at PHM 04694-10, allele Tat PHM 01811-32, allele Tat PHM01963-15, allele Cat PHM 01963-22, allele Tat PHM 05013-12, allele TatPHM 00586-10, allele A at PHM 00049-01; and a corn seed comprising ahaplotype of: allele G at PHM 00045-01, allele A at PHM 00043-01, alleleC at PHM 01963-22, and allele T at PHM 05013-12. Also of interest areplants produced by the corn seeds.

DEFINITIONS

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular embodiments,which can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting. As used in thisspecification and the appended claims, terms in the singular and thesingular forms “a”, “an” and “the”, for example, include pluralreferents unless the content clearly dictates otherwise. Thus, forexample, reference to “plant”, “the plant” or “a plant” also includes aplurality of plants; also, depending on the context, use of the term“plant” can also include genetically similar or identical progeny ofthat plant; use of the term “a nucleic acid” optionally includes, as apractical matter, many copies of that nucleic acid molecule; similarly,the term “probe” optionally (and typically) encompasses many similar oridentical probe molecules.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation. Numeric ranges recited within the specificationare inclusive of the numbers defining the range and include each integeror any non-integer fraction within the defined range. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich the invention pertains. Although any methods and materials similaror equivalent to those described herein can be used in the practice fortesting of the present invention, the preferred materials and methodsare described herein. In describing and claiming the present invention,the following terminology will be used in accordance with thedefinitions set out below.

A “plant” can be a whole plant, any part thereof, or a cell or tissueculture derived from a plant. Thus, the term “plant” can refer to anyof: whole plants, plant components or organs (e.g., leaves, stems,roots, etc.), plant tissues, seeds, plant cells, and/or progeny of thesame. A plant cell is a cell of a plant, taken from a plant, or derivedthrough culture from a cell taken from a plant.

The term “maize plant” includes whole maize plants, maize plant cells,maize plant protoplast, maize plant cell or maize tissue culture fromwhich maize plants can be regenerated, maize plant calli, maize plantclumps and maize plant cells that are intact in maize plants or parts ofmaize plants, such as maize seeds, maize cobs, maize flowers, maizecotyledons, maize leaves, maize stems, maize buds, maize roots, maizeroot tips and the like.

The term “maize” includes any member of the species Zea mays. “Maize”and “corn” are used interchangeably herein.

A “seed” is a small embryonic plant enclosed in a protective seed coat.It is the product of the ripened plant ovule generated afterfertilization.

“Germplasm” refers to genetic material of or from an individual (e.g., aplant), a group of individuals (e.g., a plant line, variety, or family),or a clone derived from a line, variety, species, or culture. Thegermplasm can be part of an organism or cell, or can be separate fromthe organism or cell. In general, germplasm provides genetic materialwith a specific molecular makeup that provides a physical foundation forsome or all of the hereditary qualities of an organism or cell culture.As used herein, germplasm includes cells, seed or tissues from which newplants may be grown, or plant parts, such as leafs, stems, pollen, orcells, that can be cultured into a whole plant.

“Variety”, when used in conjunction with plants, encompasses botanicaland cultivated plants, including inbreds and hybrids, and means a plantgrouping within a single botanical taxon of the lowest known rank, wherethe grouping can be defined by the expression of characteristicsresulting from a given genotype or combination of genotypes.

The term “inbred” means a substantially homozygous variety.

The term “hybrid” means any offspring/progeny of a cross between twogenetically unlike individuals, including a cross of two differentinbred lines.

The term “allele” refers to one of two or more different nucleotidesequences that occur at a specific locus.

An allele is “associated with” a trait when it is linked to it and whenthe presence of the allele is an indicator that the desired trait ortrait form will occur in a plant comprising the allele.

A “BAC”, or bacterial artificial chromosome, is a cloning vector derivedfrom the naturally occurring F factor of Escherichia coli. BACs canaccept large inserts of DNA sequence. In maize, a number of BACs, orbacterial artificial chromosomes, each containing a large insert ofmaize genomic DNA, have been assembled into contigs (overlappingcontiguous genetic fragments, or “contiguous DNA”).

A “favorable allele” is the allele at a particular locus that confers,or contributes to, an agronomically desirable phenotype, e.g., toleranceto GLS, or alternatively, is an allele that allows the identification ofsusceptible plants that can be removed from a breeding program orplanting. A favorable allele of a marker is a marker allele thatsegregates with the favorable phenotype, or alternatively, segregateswith susceptible plant phenotype, therefore providing the benefit ofidentifying disease-prone plants. A favorable allelic form of achromosome segment is a chromosome segment that includes a nucleotidesequence that contributes to superior agronomic performance at one ormore genetic loci physically located on the chromosome segment.

“Allele frequency” refers to the frequency (proportion or percentage) atwhich an allele is present at a locus within an individual, within aline, or within a population of lines. For example, for an allele “A”,diploid individuals of genotype “AA”, “Aa”, or “aa” have allelefrequencies of 1.0, 0.5, or 0.0, respectively. One can estimate theallele frequency within a line by averaging the allele frequencies of asample of individuals from that line. Similarly, one can calculate theallele frequency within a population of lines by averaging the allelefrequencies of lines that make up the population. For a population witha finite number of individuals or lines, an allele frequency can beexpressed as a count of individuals or lines (or any other specifiedgrouping) containing the allele.

An allele “positively” correlates with a trait when it is linked to itand when presence of the allele is an indicator that the desired traitor trait form will occur in a plant comprising the allele. An allele“negatively” correlates with a trait when it is linked to it and whenpresence of the allele is an indicator that a desired trait or traitform will not occur in a plant comprising the allele.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes).

An individual is “heterozygous” if more than one allele type is presentat a given locus (e.g., a diploid individual with one copy each of twodifferent alleles).

A special case of a heterozygous situation is where one chromosome hasan allele of a gene and the other chromosome lacks the gene, locus, orregion completely—in other words, has a deletion relative to the firstchromosome. This situation is referred to as “hemizygous”.

The term “homogeneity” indicates that members of a group have the samegenotype at one or more specific loci. In contrast, the term“heterogeneity” is used to indicate that individuals within the groupdiffer in genotype at one or more specific loci.

A “locus” is a chromosomal region where a polymorphic nucleic acid,trait determinant, gene, or marker is located. Thus, for example, a“gene locus” is a specific chromosome location in the genome of aspecies where a specific gene can be found. A locus correlated with GLStolerance denotes a region on the genome that is directly related to aphenotypically quantifiable GLS tolerance trait.

A “genetic complement” has at least one set or ploidy of alleles. Forexample, a single cross hybrid inherits two genetic complements, onefrom each inbred parent.

The term “quantitative trait locus” or “QTL” refers to a polymorphicgenetic locus with at least one allele that correlates with thedifferential expression of a phenotypic trait in at least one geneticbackground, e.g., in at least one breeding population or progeny. A QTLcan act through a single gene mechanism or by a polygenic mechanism.

The terms “marker”, “molecular marker”, “marker nucleic acid”, and“marker locus” refer to a nucleotide sequence or encoded product thereof(e.g., a protein) used as a point of reference when identifying a linkedlocus. A marker can be derived from genomic nucleotide sequence or fromexpressed nucleotide sequences (e.g., from a spliced RNA or a cDNA), orfrom an encoded polypeptide. The term also refers to nucleic acidsequences complementary to or flanking the marker sequences, such asnucleic acids used as probes or primer pairs capable of amplifying themarker sequence. A large number of maize molecular markers are known inthe art, and are published or available from various sources, such asthe Maize GDB internet resource and the Arizona Genomics Instituteinternet resource run by the University of Arizona. Similarly, numerousmethods for detecting molecular markers are well established.

A “polymorphism” is a variation in the DNA that is too common to be duemerely to new mutation (i.e. occurs at a frequency of at least 1% in apopulation). Any differentially inherited polymorphic trait (includingnucleic acid polymorphism) that segregates among progeny is a potentialmarker. The genomic variability can be of any origin, for example,insertions, deletions, duplications, repetitive elements, pointmutations, recombination events, or the presence and sequence oftransposable elements.

A “marker probe” is a nucleic acid sequence or molecule that can be usedto identify the presence of a marker locus, e.g., a nucleic acid probethat is complementary to a marker locus sequence. Alternatively, in someaspects, a marker probe refers to a probe of any type that is able todistinguish (i.e., genotype) the particular allele that is present at amarker locus. Nucleic acids are “complementary” when they specificallyhybridize in solution, e.g., according to Watson-Crick base pairingrules.

A “marker locus” is a locus that can be used to track the presence of asecond linked locus, e.g., a linked locus that encodes or contributes toexpression of a phenotypic trait. For example, a marker locus can beused to monitor segregation of alleles at a locus, such as a QTL, thatare genetically or physically linked to the marker locus.

A “marker allele”, alternatively an “allele of a marker locus”, is oneof a plurality of polymorphic nucleotide sequences found at a markerlocus in a population that is polymorphic for the marker locus. In someaspects, the present invention provides marker loci correlating withtolerance to GLS in maize. Each of the identified markers is expected tobe in close physical and genetic proximity (resulting in physical and/orgenetic linkage) to a genetic element, e.g., a QTL, that contributes toGLS tolerance.

“Genetic markers” are nucleic acids that are polymorphic in a populationand where the alleles of which can be detected and distinguished by oneor more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and thelike. The term also refers to nucleic acid sequences complementary tothe genomic sequences, such as nucleic acids used as probes. The term“Genetic Marker” can refer to any type of nucleic acid based marker,including but not limited to, Restriction Fragment Length Polymorphism(RFLP), Simple Sequence Repeat (SSR), Random Amplified Polymorphic DNA(RAPD), Cleaved Amplified Polymorphic Sequences (CAPS) (Rafalski andTingey, 1993, Trends in Genetics 9:275-280), Amplified Fragment LengthPolymorphism (AFLP) (Vos et al, 1995, Nucleic Acids Res. 23:4407-4414),Single Nucleotide Polymorphism (SNP) (Brookes, 1999, Gene 234:177-186),Sequence Characterized Amplified Region (SCAR) (Paran and Michelmore,1993, Theor. Appl. Genet. 85:985-993), Sequence Tagged Site (STS)(Onozaki et al., 2004, Euphytica 138:255-262), Single StrandedConformation Polymorphism (SSCP) (Orita et al., 1989, Proc Natl Acad SciUSA 86:2766-2770), Inter-Simple Sequence Repeat (ISSR) (Blair et al.,1999, Theor. Appl. Genet. 98:780-792), Inter-Retrotransposon AmplifiedPolymorphism (IRAP), Retrotransposon-Microsatellite AmplifiedPolymorphism (REMAP) (Kalendar et al., 1999, Theor. Appl. Genet.98:704-711), an RNA cleavage product (such as a Lynx tag), and the like.

The term “indel” refers to an insertion or deletion, wherein one linemay be referred to as having an insertion relative to a second line, orthe second line may be referred to as having a deletion relative to thefirst line.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by methods well-established in the art. Theseinclude, e.g., PCR-based sequence specific amplification methods,detection of restriction fragment length polymorphisms (RFLP), detectionof isozyme markers, detection of polynucleotide polymorphisms by allelespecific hybridization (ASH), detection of amplified variable sequencesof the plant genome, detection of self-sustained sequence replication,detection of simple sequence repeats (SSRs), detection of singlenucleotide polymorphisms (SNPs), or detection of amplified fragmentlength polymorphisms (AFLPs). Well established methods are also know forthe detection of expressed sequence tags (ESTs) and SSR markers derivedfrom EST sequences and randomly amplified polymorphic DNA (RAPD).Methods of mapping a gene via cleaved amplified polymorphic sequences(CAPS) are also well-known (see, e.g., Konieczny & Ausubel, Plant J.4:403-10 (1993)).

“Marker assisted selection” (or MAS) is a process by which phenotypesare selected based on marker genotypes.

“Marker assisted counter-selection” is a process by which markergenotypes are used to identify plants that will not be selected,allowing them to be removed from a breeding program or planting.

A “reference sequence” is a defined sequence used as a basis forsequence comparison. The reference sequence is obtained by genotyping anumber of lines at the locus, aligning the nucleotide sequences in asequence alignment program (e.g. Sequencher), and then obtaining theconsensus sequence of the alignment.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form. The lociare genetic landmarks, or markers, and for each genetic map, distancesbetween markers are measured by the recombination frequencies betweenthem. A genetic map is a product of the mapping population, types ofmarkers used, and the polymorphic potential of each marker betweendifferent populations. The order and genetic distances between loci candiffer from one genetic map to another. For example, 10 cM on theinternally derived genetic map (also referred to herein as “PHB” forPioneer Hi-Bred) is roughly equivalent to 25-30 cM on the IBM2 2005neighbors frame map (a high resolution map available on maizeGDB).However, information can be correlated from one map to another using ageneral framework of common markers. One of ordinary skill in the artcan use the framework of common markers to identify the positions ofmarkers and other loci of interest on each individual genetic map.

“Genetic mapping” is the process of defining the linkage relationshipsof loci through the use of genetic markers, populations segregating forthe markers, and standard genetic principles of recombination frequency.A “genetic map location” is a location on a genetic map relative tosurrounding genetic markers on the same linkage group where a specifiedmarker can be found within a given species.

A “physical map” of the genome refers to absolute distances (forexample, measured in base pairs or isolated and overlapping contiguousgenetic fragments, e.g., contigs). A physical map of the genome does nottake into account the genetic behavior (e.g., recombination frequencies)between different points on the physical map.

“Recombination” is an exchange of segments of homologous chromosomesduring meiosis whereby linked genes become recombined; also refers tothe product of such exchange.

A “genetic recombination frequency” is the frequency of a crossing overevent (recombination) between two genetic loci. Recombination frequencycan be observed by following the segregation of markers and/or traitsfollowing meiosis. A genetic recombination frequency can be expressed incentimorgans (cM), where one cM is the distance between two geneticmarkers that show a 1% recombination frequency (i.e., a crossing-overevent occurs between those two markers once in every 100 celldivisions).

“Genome” refers to the total DNA, or the entire set of genes, carried bya chromosome or chromosome set.

As used herein, the term “linkage” is used to describe the degree withwhich one marker locus is “associated with” another marker locus or someother locus (for example, a tolerance locus). The closer two marker locilie on the same chromosome, the more closely they will be associated ingametes and the more often they will appear together; marker loci thatare very close are essentially never separated because it is extremelyunlikely that a crossover point will occur between them.

As used herein, “linkage equilibrium” describes a situation where twomarkers independently segregate, i.e., sort among progeny randomly.Markers that show linkage equilibrium are considered unlinked (whetheror not they lie on the same chromosome).

The term “linkage disequilibrium” refers to a non-random segregation ofgenetic loci or traits (or both). In either case, linkage disequilibriumimplies that the relevant loci are within sufficient physical proximityalong a length of a chromosome so that they segregate together withgreater than random (i.e., non-random) frequency (in the case ofco-segregating traits, the loci that underlie the traits are insufficient proximity to each other). Markers that show linkagedisequilibrium are considered linked.

Linkage disequilibrium is most commonly assessed using the measure r²,which is calculated using the formula described by Hill, W. G. andRobertson, A, Theor. Appl. Genet. 38:226-231(1968). When r²=1, completeLD exists between the two marker loci, meaning that the markers have notbeen separated by recombination and have the same allele frequency.Values for r² above ⅓ indicate sufficiently strong LD to be useful formapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)).Hence, alleles are in linkage disequilibrium when r² values betweenpairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, or 1.0.

As used herein, the linkage relationship between a molecular marker anda phenotype is given as a “probability” or “adjusted probability”. Theprobability value is the statistical likelihood that the particularcombination of a phenotype and the presence or absence of a particularmarker allele is random. Thus, the lower the probability score, thegreater the likelihood that a phenotype and a particular marker willco-segregate. In some aspects, the probability score is considered“significant” or “nonsignificant”. In some embodiments, a probabilityscore of 0.05 (p=0.05, or a 5% probability) of random assortment isconsidered a significant indication of co-segregation. However, thepresent invention is not limited to this particular standard, and anacceptable probability can be any probability of less than 50% (p=0.5).For example, a significant probability can be less than 0.25, less than0.20, less than 0.15, or less than 0.1.

The term “physically linked” is sometimes used to indicate that twoloci, e.g., two marker loci, are physically present on the samechromosome.

Advantageously, the two linked loci are located in close proximity suchthat recombination between homologous chromosome pairs does not occurbetween the two loci during meiosis with high frequency, e.g., such thatlinked loci co-segregate at least about 90% of the time, e.g., 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time.

The phrase “closely linked”, in the present application, means thatrecombination between two linked loci occurs with a frequency of equalto or less than about 10% (i.e., are separated on a genetic map by notmore than 10 cM). Put another way, the closely linked loci co-segregateat least 90% of the time. Marker loci are especially useful in thepresent invention when they demonstrate a significant probability ofco-segregation (linkage) with a desired trait (e.g., pathogenictolerance). For example, in some aspects, these markers can be termedlinked QTL markers. In other aspects, especially useful molecularmarkers are those markers that are linked or closely linked.

In some aspects, linkage can be expressed as any desired limit or range.For example, in some embodiments, two linked loci are two loci that areseparated by less than 50 cM map units. In other embodiments, linkedloci are two loci that are separated by less than 40 cM. In otherembodiments, two linked loci are two loci that are separated by lessthan 30 cM. In other embodiments, two linked loci are two loci that areseparated by less than 25 cM. In other embodiments, two linked loci aretwo loci that are separated by less than 20 cM. In other embodiments,two linked loci are two loci that are separated by less than 15 cM. Insome aspects, it is advantageous to define a bracketed range of linkage,for example, between 10 and 20 cM, or between 10 and 30 cM, or between10 and 40 cM.

The more closely a marker is linked to a second locus, the better anindicator for the second locus that marker becomes. Thus, in oneembodiment, closely linked loci such as a marker locus and a secondlocus display an inter-locus recombination frequency of 10% or less,preferably about 9% or less, still more preferably about 8% or less, yetmore preferably about 7% or less, still more preferably about 6% orless, yet more preferably about 5% or less, still more preferably about4% or less, yet more preferably about 3% or less, and still morepreferably about 2% or less. In highly preferred embodiments, therelevant loci display a recombination frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Two loci that are localized to thesame chromosome, and at such a distance that recombination between thetwo loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to beclosely linked to each other. In some cases, two different markers canhave the same genetic map coordinates. In that case, the two markers arein such close proximity to each other that recombination occurs betweenthem with such low frequency that it is undetectable.

When referring to the relationship between two genetic elements, such asa genetic element contributing to tolerance and a closely linked marker,“coupling” phase linkage indicates the state where the “favorable”allele at the tolerance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest isphysically linked with an “unfavorable” allele at the linked markerlocus, and the two “favorable” alleles are not inherited together (i.e.,the two loci are “out of phase” with each other).

As used herein, the terms “chromosome interval”, “chromosomal interval”,“chromosome segment”, or “chromosomal segment” designate a contiguouslinear span of genomic DNA that resides in planta on a singlechromosome, usually defined with reference to two markers defining theend points of the chromosomal interval. The genetic elements or geneslocated on a single chromosome interval are physically linked. The sizeof a chromosome interval is not particularly limited.

In some aspects, for example in the context of the present invention,generally the genetic elements located within a single chromosomeinterval are also genetically linked, typically within a geneticrecombination distance of, for example, less than or equal to 20 cM, oralternatively, less than or equal to 10 cM. That is, two geneticelements within a single chromosome interval undergo recombination at afrequency of less than or equal to 20% or 10%.

In one aspect, any marker of the invention is linked (genetically andphysically) to any other marker that is at or less than 50 cM distant.In another aspect, any marker of the invention is closely linked(genetically and physically) to any other marker that is in closeproximity, e.g., at or less than 10 cM distant. Two closely linkedmarkers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2,1, 0.75, 0.5 or 0.25 cM or less from each other.

The phrase “Gray Leaf Spot” or “GLS” refers to a cereal disease causedby the fungal pathogen Cercospora zeae-maydis, which characteristicallyproduces long, rectangular, grayish-tan leaf lesions which run parallelto the leaf vein.

“Newly conferred tolerance” or “enhanced tolerance” in a maize plant toGLS is an indication that the maize plant is less affected with respectto yield and/or survivability or other relevant agronomic measures, uponintroduction of the causative agents of that disease, e.g., Cercosporazeae-maydis. Tolerance is a relative term, indicating that the infectedplant produces better yield of maize than another, similarly treated,more susceptible plant. That is, the conditions cause a reduced decreasein maize survival and/or yield in a tolerant maize plant, as compared toa susceptible maize plant.

One of skill will appreciate that maize plant tolerance to GLS varieswidely, can represent a spectrum of more tolerant or less tolerantphenotypes, and can vary depending on the severity of the infection.However, by simple observation, one of skill can determine the relativetolerance or susceptibility of different plants, plant lines or plantfamilies to GLS, and furthermore, will also recognize the phenotypicgradations of “tolerant”. For example, a 1 to 9 visual rating indicatingthe tolerance to GLS can be used. A higher score indicates a higherresistance. Data should be collected only when sufficient selectionpressure exists in the experiment measured.

The term “crossed” or “cross” in the context of this invention means thefusion of gametes via pollination to produce progeny (e.g., cells, seedsor plants). The term encompasses both sexual crosses (the pollination ofone plant by another) and selfing (self-pollination, e.g., when thepollen and ovule are from the same plant). The term “crossing” refers tothe act of fusing gametes via pollination to produce progeny.

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. For example,introgression of a desired allele at a specified locus can betransmitted to at least one progeny via a sexual cross between twoparents of the same species, where at least one of the parents has thedesired allele in its genome. Alternatively, for example, transmissionof an allele can occur by recombination between two donor genomes, e.g.,in a fused protoplast, where at least one of the donor protoplasts hasthe desired allele in its genome. The desired allele can be, e.g., aselected allele of a marker, a QTL, a transgene, or the like. In anycase, offspring comprising the desired allele can be repeatedlybackcrossed to a line having a desired genetic background and selectedfor the desired allele, to result in the allele becoming fixed in aselected genetic background.

The process of “introgressing” is often referred to as “backcrossing”when the process is repeated two or more times.

A “backcross conversion” is a product of introgression of a locus ortrait into a variety by backcrossing.

“Backcrossing” refers to the process whereby hybrid progeny arerepeatedly crossed back to one of the parents. In a backcrossing scheme,the “donor” parent refers to the parental plant with the desired gene orlocus to be introgressed. The “recipient” parent (used one or moretimes) or “recurrent” parent (used two or more times) refers to theparental plant into which the gene or locus is being introgressed. Forexample, see Ragot, M. et al. (1995) Marker-assisted backcrossing: apractical example, in Techniques et Utilisations des MarqueursMoleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al.,(1994) Marker-assisted Selection in Backcross Breeding, Analysis ofMolecular Marker Data, pp. 41-43. The initial cross gives rise to the F1generation; the term “BC1” then refers to the second use of therecurrent parent, “BC2” refers to the third use of the recurrent parent,and so on.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendents that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor.

An “ancestral line” is a parent line used as a source of genes e.g., forthe development of elite lines. An “ancestral population” is a group ofancestors that have contributed the bulk of the genetic variation thatwas used to develop elite lines. “Progeny” are the descendants ofancestors, and may be separated from their ancestors by many generationsof breeding. For example, elite lines are the progeny of theirancestors. A “pedigree structure” defines the relationship between aprogeny and each ancestor that gave rise to that descendant. A pedigreestructure can span one or more generations, describing relationshipsbetween the progeny and its parents, grand parents, great-grand parents,etc.

An “elite line” or “elite strain” is an agronomically superior line thathas resulted from many cycles of breeding and selection for superioragronomic performance. Numerous elite lines are available and known tothose of skill in the art of maize breeding. An “elite population” is anassortment of elite individuals or lines that can be used to representthe state of the art in terms of agronomically superior genotypes of agiven crop species, such as maize. Similarly, an “elite germplasm” orelite strain of germplasm is an agronomically superior germplasm,typically derived from and/or capable of giving rise to a plant withsuperior agronomic performance, such as an existing or newly developedelite line of maize.

In contrast, an “exotic maize strain” or an “exotic maize germplasm” isa strain or germplasm derived from maize that does not belong to anavailable elite maize line or strain of germplasm. In the context of across between two maize plants or strains of germplasm, an exoticgermplasm is not closely related by descent to the elite germplasm withwhich it is crossed. Most commonly, the exotic germplasm is not derivedfrom any known elite line of maize, but rather is selected to introducenovel genetic elements (typically novel alleles) into a breedingprogram.

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. Typical amplification methodsinclude various polymerase based replication methods, including thepolymerase chain reaction (PCR), ligase mediated methods such as theligase chain reaction (LCR), and RNA polymerase based amplification(e.g., by transcription) methods.

An “amplicon” is an amplified nucleic acid, e.g., a nucleic acid that isproduced by amplifying a template nucleic acid by any availableamplification method (e.g., PCR, LCR, transcription, or the like).

A “genomic nucleic acid” is a nucleic acid that corresponds in sequenceto a heritable nucleic acid in a cell. Common examples include nucleargenomic DNA and amplicons thereof. A genomic nucleic acid is, in somecases, different from a spliced RNA, or a corresponding cDNA, in thatthe spliced RNA or cDNA is processed, e.g., by the splicing machinery,to remove introns. Genomic nucleic acids optionally comprisenon-transcribed (e.g., chromosome structural sequences, promoterregions, or enhancer regions) and/or non-translated sequences (e.g.,introns), whereas spliced RNA/cDNA typically do not have non-transcribedsequences or introns. A “template nucleic acid” is a nucleic acid thatserves as a template in an amplification reaction (e.g., a polymerasebased amplification reaction such as PCR, a ligase mediatedamplification reaction such as LCR, a transcription reaction, or thelike). A template nucleic acid can be genomic in origin, oralternatively, can be derived from expressed sequences, e.g., a cDNA oran EST.

“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and“nucleic acid fragment” are used interchangeably and refer to a polymerof RNA or DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. A “nucleotide” is amonomeric unit from which DNA or RNA polymers are constructed, andconsists of a purine or pyrimidine base, a pentose, and a phosphoricacid group. Nucleotides (usually found in their 5′-monophosphate form)are referred to by their single letter designation as follows: “A” foradenylate or deoxyadenylate (for RNA or DNA, respectively), “C” forcytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U”for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y”for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” forinosine, and “N” for any nucleotide.

An “exogenous nucleic acid” is a nucleic acid that is not native to aspecified system (e.g., a germplasm, plant, or variety), with respect tosequence, genomic position, or both. As used herein, the terms“exogenous” or “heterologous” as applied to polynucleotides orpolypeptides typically refers to molecules that have been artificiallysupplied to a biological system (e.g., a plant cell, a plant gene, aparticular plant species or variety or a plant chromosome under study)and are not native to that particular biological system. The terms canindicate that the relevant material originated from a source other thana naturally occurring source, or can refer to molecules having anon-natural configuration, genetic location or arrangement of parts.

In contrast, for example, a “native” or “endogenous” gene is a gene thatdoes not contain nucleic acid elements encoded by sources other than thechromosome or other genetic element on which it is normally found innature. An endogenous gene, transcript or polypeptide is encoded by itsnatural chromosomal locus, and not artificially supplied to the cell.

The term “recombinant” in reference to a nucleic acid or polypeptideindicates that the material (e.g., a recombinant nucleic acid, gene,polynucleotide, or polypeptide) has been altered by human intervention.Generally, the arrangement of parts of a recombinant molecule is not anative configuration, or the primary sequence of the recombinantpolynucleotide or polypeptide has in some way been manipulated. Thealteration to yield the recombinant material can be performed on thematerial within or removed from its natural environment or state. Forexample, a naturally occurring nucleic acid becomes a recombinantnucleic acid if it is altered, or if it is transcribed from DNA whichhas been altered, by means of human intervention performed within thecell from which it originates. A gene sequence open reading frame isrecombinant if that nucleotide sequence has been removed from itsnatural context and cloned into any type of artificial nucleic acidvector. Protocols and reagents to produce recombinant molecules,especially recombinant nucleic acids, are common and routine in the art.In one embodiment, an artificial chromosome can be created and insertedinto maize plants by any method known in the art (e.g., direct transferprocesses, such as, e.g., PEG-induced DNA uptake, protoplast fusion,microinjection, electroporation, and microprojectile bombardment). Anartificial chromosome is a piece of DNA that can stably replicate andsegregate alongside endogenous chromosomes. It has the capacity toaccommodate and express heterologous genes inserted therein. Integrationof heterologous DNA into the megareplicator region (primary replicationinitiation site of centromeres) or in close proximity thereto, initiatesa large-scale amplification of megabase-size chromosomal segments, whichleads to de novo chromosome formation. See, e.g., U.S. Pat. No.6,077,697, incorporated herein by reference.

The term recombinant can also refer to an organism that harborsrecombinant material, e.g., a plant that comprises a recombinant nucleicacid is considered a recombinant plant. In some embodiments, arecombinant organism is a transgenic organism.

The term “introduced” when referring to translocating a heterologous orexogenous nucleic acid into a cell refers to the incorporation of thenucleic acid into the cell using any methodology. The term encompassessuch nucleic acid introduction methods as “transfection”,“transformation”, and “transduction”.

As used herein, the term “vector” is used in reference to polynucleotideor other molecules that transfer nucleic acid segment(s) into a cell.The term “vehicle” is sometimes used interchangeably with “vector”. Avector optionally comprises parts which mediate vector maintenance andenable its intended use (e.g., sequences necessary for replication,genes imparting drug or antibiotic resistance, a multiple cloning site,or operably linked promoter/enhancer elements which enable theexpression of a cloned gene). Vectors are often derived from plasmids,bacteriophages, or plant or animal viruses. A “cloning vector” or“shuttle vector” or “subcloning vector” contains operably linked partsthat facilitate subcloning steps (e.g., a multiple cloning sitecontaining multiple restriction endonuclease sites).

The term “expression vector” as used herein refers to a vectorcomprising operably linked polynucleotide sequences that facilitateexpression of a coding sequence in a particular host organism (e.g., abacterial expression vector or a plant expression vector).Polynucleotide sequences that facilitate expression in prokaryotestypically include, e.g., a promoter, an operator (optional), and aribosome binding site, often along with other sequences. Eukaryoticcells can use promoters, enhancers, termination and polyadenylationsignals, and other sequences that are generally different from thoseused by prokaryotes.

The term “transgenic plant” refers to a plant that comprises within itscells a heterologous polynucleotide. Generally, the heterologouspolynucleotide is stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant expression cassette. “Transgenic” is used herein to refer toany cell, cell line, callus, tissue, plant part or plant, the genotypeof which has been altered by the presence of heterologous nucleic acidincluding those transgenic organisms or cells initially so altered, aswell as those created by crosses or asexual propagation from the initialtransgenic organism or cell. The term “transgenic” as used herein doesnot encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods (e.g.,crosses) or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

“Positional cloning” is a cloning procedure in which a target nucleicacid is identified and isolated by its genomic proximity to markernucleic acid. For example, a genomic nucleic acid clone can include partor all of two more chromosomal regions that are closely linked to oneanother. If a marker can be used to identify the genomic nucleic acidclone from a genomic library, standard methods such as sub-cloning orsequencing can be used to identify and/or isolate subsequences of theclone that are located near the marker.

A specified nucleic acid is “derived from” a given nucleic acid when itis constructed using the given nucleic acid's sequence, or when thespecified nucleic acid is constructed using the given nucleic acid. Forexample, a cDNA or EST is derived from an expressed mRNA.

The term “genetic element” or “gene” refers to a heritable sequence ofDNA, i.e., a genomic sequence, with functional significance. The term“gene” can also be used to refer to, e.g., a cDNA and/or an mRNA encodedby a genomic sequence, as well as to that genomic sequence.

The term “genotype” is the genetic constitution of an individual (orgroup of individuals) at one or more genetic loci, as contrasted withthe observable trait (the phenotype). Genotype is defined by theallele(s) of one or more known loci that the individual has inheritedfrom its parents. The term genotype can be used to refer to anindividual's genetic constitution at a single locus, at multiple loci,or, more generally, the term genotype can be used to refer to anindividual's genetic make-up for all the genes in its genome.

A “haplotype” is the genotype of an individual at a plurality of geneticloci. Typically, the genetic loci described by a haplotype arephysically and genetically linked, i.e., on the same chromosome segment.The term “haplotype” can refer to polymorphisms at a particular locus,such as a single marker locus, or polymorphisms at multiple loci along achromosomal segment. The former can also be referred to as “markerhaplotypes” or “marker alleles”, while the latter can be referred to as“long-range haplotypes”.

The terms “phenotype”, or “phenotypic trait” or “trait” refers to one ormore trait of an organism. The phenotype can be observable to the nakedeye, or by any other means of evaluation known in the art, e.g.,microscopy, biochemical analysis, genomic analysis, or an assay for aparticular disease tolerance. In some cases, a phenotype is directlycontrolled by a single gene or genetic locus, i.e., a “single genetrait”. In other cases, a phenotype is the result of several genes.

A “molecular phenotype” is a phenotype detectable at the level of apopulation of (one or more) molecules. Such molecules can be nucleicacids such as genomic DNA or RNA, proteins, or metabolites. For example,a molecular phenotype can be an expression profile for one or more geneproducts, e.g., at a specific stage of plant development, in response toan environmental condition or stress, etc. Expression profiles aretypically evaluated at the level of RNA or protein, e.g., on a nucleicacid array or “chip” or using antibodies or other binding proteins.

A “representative sample” is a sample that encompasses the relevantcomposition and characteristics of the population sampled.

A “centromere” is the single site on each chromosome for kinetochoreassembly and proper chromosome segregation in mitosis and meiosis.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. For example, yield ofmaize is commonly measured in bushels of seed per acre or metric tons ofseed per hectare per season. Yield is affected by both genetic andenvironmental factors. “Agronomics”, “agronomic traits”, and “agronomicperformance” refer to the traits (and underlying genetic elements) of agiven plant variety that contribute to yield over the course of growingseason. Individual agronomic traits include emergence vigor, vegetativevigor, stress tolerance, disease resistance or tolerance, herbicideresistance, branching, flowering, seed set, seed size, seed density,standability, threshability, and the like. Yield is, therefore, thefinal culmination of all agronomic traits.

A “set” of markers or probes refers to a collection or group of markersor probes, or the data derived therefrom, used for a common purpose,e.g., identifying maize plants with a desired trait (e.g., tolerance toGLS). Frequently, data corresponding to the markers or probes, or dataderived from their use, is stored in an electronic medium. While each ofthe members of a set possess utility with respect to the specifiedpurpose, individual markers selected from the set as well as subsetsincluding some, but not all, of the markers are also effective inachieving the specified purpose.

A “genetic profile” is an identification and characterization ofsequences diagnostic for a particular trait or locus.

A “look up table” is a table that correlates one form of data toanother, or one or more forms of data with a predicted outcome that thedata is relevant to. For example, a look up table can include acorrelation between allele data and a predicted trait that a plantcomprising a given allele is likely to display. These tables can be, andtypically are, multidimensional, e.g., taking multiple alleles intoaccount simultaneously, and, optionally, taking other factors intoaccount as well, such as genetic background, e.g., in making a traitprediction.

A “contig” refers to a set of overlapping DNA segments derived from asingle genetic source. A contig map depicts the relative order of alinked library of contigs representing an extended chromosome segment. A“public contig” is a publicly available set of overlapping DNA segmentsderived from a single genetic source. Examples of public sources includethe Maize Mapping Project (University of Missouri—Columbia, Universityof Georgia, and University of Arizona), the Arizona Genomics Institute,the MaizeGDB website, and the Maize Sequence website. Sequencealignments or contigs may also be used to find sequences upstream ordownstream of the specific markers listed herein. These new sequences,close to the markers described herein, are then used to discover anddevelop functionally equivalent markers. For example, different physicaland/or genetic maps are aligned to locate equivalent markers notdescribed within this disclosure but that are within similar regions.These maps may be within the maize species, or even across other speciesthat have been genetically or physically aligned with maize, such asrice, wheat, barley, or sorghum.

Sequence alignments and percent similarity calculations may bedetermined using the Megalign program of the LASARGENE bioinformaticscomputing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of thesequences are performed using the Clustal method of alignment (Higginsand Sharp, CABIOS 5:151-153 (1989)) with the default parameters (GAPPENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwisealignments and calculation of percent identity of protein sequencesusing the Clustal method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 andDIAGONALS SAVED=5. For nucleic acids these parameters are GAPPENALTY=10, GAP LENGTH PENALTY=10, KTUPLE=2, GAP PENALTY=5, WINDOW=4,and DIAGONALS SAVED=4. A “substantial portion” of an amino acid ornucleotide sequence comprises enough of the amino acid sequence of apolypeptide or the nucleotide sequence of a gene to afford putativeidentification of that polypeptide or gene, either by manual evaluationof the sequence by one skilled in the art, or by computer-automatedsequence comparison and identification using algorithms such as BLAST(Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1993)) and GappedBlast (Altschul, S. F. et al., Nucleic Acids Res. 25:3389-3402 (1997)).BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleotide sequences homologous to anucleotide sequence encoding a protein of the embodiments. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to a protein orpolypeptide of the embodiments. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul et al. (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.,BLASTN for nucleotide sequences, BLASTX for proteins) can be used.Alignment may also be performed manually by inspection.

A “computer readable medium” is an information storage media that can beaccessed by a computer using an available or custom interface. Examplesinclude memory (e.g., ROM, RAM, or flash memory), optical storage media(e.g., CD-ROM), magnetic storage media (computer hard drives, floppydisks, etc.), punch cards, and many others that are commerciallyavailable. Information can be transmitted between a system of interestand the computer, or to or from the computer and the computer readablemedium for storage or access of stored information. This transmissioncan be an electrical transmission, or can be made by other availablemethods, such as an IR link, a wireless connection, or the like.

“System instructions” are instruction sets that can be partially orfully executed by the system. Typically, the instruction sets arepresent as system software.

BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCES

The invention can be more fully understood from the following detaileddescription and the accompanying drawings and Sequence Listing whichform a part of this application. The Sequence Listing contains the oneletter code for nucleotide sequence characters and the three lettercodes for amino acids as defined in conformity with the IUPAC-IUBMBstandards described in Nucleic Acids Research 13:3021-3030 (1985) and inthe Biochemical Journal 219 (No. 2): 345-373 (1984), which are hereinincorporated by reference in their entirety. The symbols and format usedfor nucleotide and amino acid sequence data comply with the rules setforth in 37 C.F.R. §1.822.

FIG. 1 shows the final location of the PHJEP GLS QTL on the IBM2 2004neighbors chromosome 4 map. Distances are in cM.

FIG. 2 shows a close-up of the GLS QTL on the IBM2 2004 neighborschromosome 4 map after the second round of mapping. Boxed markers wereused in the second round of QTL mapping. Arrow shows QTL region afterthis second round of QTL mapping. Black-filled box shows final QTLlocation after fine mapping.

FIGS. 3A and 3B show the GLS QTL map location after fine mapping.

FIGS. 4A-F show the physical map arrangement of sequenced BACs (obtainedfrom the Maize Genome Browser, which is publicly available on theinternet) in the chromosome 4 region containing the GLS QTL.

FIG. 5 shows introgression of the GLS QTL into PHN46. Dark grayindicates PH14T origin. Horizontal lines indicate PHN46 origin. Diagonallines indicate region of recombination. *Location for UMC1299 taken fromIBM2 Neighbors frame map—concordant with physical map location.

FIG. 6 shows levels of disease resistance to GLS in PHN46 (left), PH14T(middle) and PHJEP (right).

FIG. 7 shows levels of disease resistance to GLS in A) a Pioneer hybridwith the QTL introgression, created by a cross between inbreds PHP38 andPHJEP, and in B) Pioneer hybrid 3394 with no QTL introgression.

FIG. 8 shows further introgression of the GLS QTL into elite materials.Dark gray indicates PH14T origin. Horizontal lines indicate PHN46origin. Diagonal lines indicate recombination between PHJEP and newelite germplasm. Dots indicate recombination between PH14T and PHN46.Unshaded indicates new elite germplasm PHVNV, PHEHG, PHW3Y, PHEWB, orPHWRC.

FIG. 9 provides a table listing genomic and SSR markers, including thosemarkers that demonstrated linkage disequilibrium with the GLS tolerancephenotype (directly or by extrapolation from the genetic map). The tableprovides the sequences of the left and right PCR primers used in the SSRmarker locus genotyping analysis. Also shown is the number ofnucleotides in the tandem repeating element in the SSR.

FIG. 10 provides a table listing the SNP markers that demonstratedlinkage disequilibrium with the GLS tolerance phenotype. The tableprovides the sequences of the PCR primers used to generate aSNP-containing amplicon.

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R.§1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. §1.822.

SEQ ID NO:1 and SEQ ID NO:2 are the primers designed to amplify BAC endbacm2.pk027.h10.f.

SEQ ID NO:3 and SEQ ID NO:4 are the primers designed to amplify BAC endbacm.pk098.d7.

SEQ ID NO:5 and SEQ ID NO:6 are the primers designed to amplify BAC endbacm.pk106.j3.

SEQ ID NO:7 and SEQ ID NO:8 are the primers designed to amplify BAC endbacm.pk018.h15.

SEQ ID NO:9 and SEQ ID NO:10 are the primers designed to amplify BAC endbacm.pk040.o17. The primer pair represents a CAPs marker referred toherein as PHM 00045.

SEQ ID NO:11 and SEQ ID NO:12 are the primers designed to amplify BACend bacm2.pk065.b22.f.

SEQ ID NO:13 and SEQ ID NO:14 are the primers designed to amplify BACend bacb.pk0333.o19. The primer pair represents a CAPs marker referredto herein as PHM 00043.

SEQ ID NO:15 and SEQ ID NO:16 are the primers designed to amplify BACend bacc.pk0267.m12.f.

SEQ ID NO:17 and SEQ ID NO:18 are the primers designed to amplify theEST overgo probe cl33021_(—)1.

SEQ ID NO:19 and SEQ ID NO:20 are the primers designed to amplify BACend bacb.pk0241.h17.f.

SEQ ID NO:21 and SEQ ID NO:22 are the primers designed to amplify clonechp2.pk0007.d2.

SEQ ID NO:23 and SEQ ID NO:24 are the primers designed to amplify BACend bacc.pk0530.f13.f.

SEQ ID NO:25 and SEQ ID NO:26 are the primers designed to clonep0094.csstg88.

SEQ ID NO:27 and SEQ ID NO:28 are the primers designed to amplify BACend bacb.pk0269.n19.

SEQ ID NO:29 and SEQ ID NO:30 are the primers designed to amplify BACend bacb.pk0009.b21.f.

SEQ ID NO:31 and SEQ ID NO:32 are the primers designed to amplify BACend bacb.pk0117.i09.f.

SEQ ID NO:33 and SEQ ID NO:34 are the primers designed to amplify BACend bacc.pk0280.n12.

SEQ ID NO:35 and SEQ ID NO:36 are the primers designed to amplify BACend bacb.pk0219.j20.

SEQ ID NO:37 and SEQ ID NO:38 are the primers designed to amplify BACend bacc.pk0132.b16.f.

SEQ ID NO:39 and SEQ ID NO:40 are the primers designed to amplify BACend bacb.pk0221.o22.

SEQ ID NO:41 and SEQ ID NO:42 are the primers designed to amplify BACend bacb.pk0544.j18.

SEQ ID NO:43 and SEQ ID NO:44 are the primers designed to amplify BACend bacb.pk0540.c18.f.

SEQ ID NO:45 and SEQ ID NO:46 are the primers designed to amplify BACend bacm.pk022.b8. The primer pair represents a CAPs marker referred toherein as PHM 00049.

SEQ ID NO:47 and SEQ ID NO:48 are the primers for marker locus PHM 7245.

SEQ ID NO:49 is the annotated nucleotide sequence of a putative R geneof the type classified as an LRR-like protein kinase.

SEQ ID NO:50 is the amino acid sequence of the protein encoded by SEQ IDNO:49.

SEQ ID NO:51 is the sequence of the PHM 15534-13 forward primer.

SEQ ID NO:52 is the sequence of the PHM 15534-13 reverse primer.

SEQ ID NO:53 is the sequence of the PHM 15534-13 probe 1.

SEQ ID NO:54 is the sequence of the PHM 15534-13 probe 2.

SEQ ID NO:55 is the sequence of the PHM 15534 reference sequence.

SEQ ID NO:56 is the sequence of the PHM 04694-10 forward primer.

SEQ ID NO:57 is the sequence of the PHM 04694-10 reverse primer.

SEQ ID NO:58 is the sequence of the PHM 04694-10 probe 1.

SEQ ID NO:59 is the sequence of the PHM 04694-10 probe 2.

SEQ ID NO:60 is the sequence of the PHM 04694-10 reference sequence.

SEQ ID NO:61 is the sequence of the PHM 01811-32 forward primer.

SEQ ID NO:62 is the sequence of the PHM 01811-32 reverse primer.

SEQ ID NO:63 is the sequence of the PHM 01811-32 probe 1.

SEQ ID NO:64 is the sequence of the PHM 01811-32 probe 2.

SEQ ID NO:65 is the sequence of the PHM 01811-32 reference sequence.

SEQ ID NO:66 is the sequence of the PHM 01963-15 forward primer.

SEQ ID NO:67 is the sequence of the PHM 01963-15 reverse primer.

SEQ ID NO:68 is the sequence of the PHM 01963-15 probe 1.

SEQ ID NO:69 is the sequence of the PHM 01963-15 probe 2.

SEQ ID NO:70 is the sequence of the PHM 01963-15 reference sequence.

SEQ ID NO:71 is the sequence of the PHM 01963-22 forward primer.

SEQ ID NO:72 is the sequence of the PHM 01963-22 reverse primer.

SEQ ID NO:73 is the sequence of the PHM 01963-22 probe 1.

SEQ ID NO:74 is the sequence of the PHM 01963-22 probe 2.

SEQ ID NO:75 is the sequence of the PHM 01963-22 reference sequence.

SEQ ID NO:76 is the sequence of the PHM 05013-12 forward primer.

SEQ ID NO:77 is the sequence of the PHM 05013-12 reverse primer.

SEQ ID NO:78 is the sequence of the PHM 05013-12 probe 1.

SEQ ID NO:79 is the sequence of the PHM 05013-12 probe 2.

SEQ ID NO:80 is the sequence of the PHM 05013-12 reference sequence.

SEQ ID NO:81 is the sequence of the PHM 00586-10 forward primer.

SEQ ID NO:82 is the sequence of the PHM 00586-10 reverse primer.

SEQ ID NO:83 is the sequence of the PHM 00586-10 probe 1.

SEQ ID NO:84 is the sequence of the PHM 00586-10 probe 2.

SEQ ID NO:85 is the sequence of the PHM 00586-10 reference sequence.

SEQ ID NO:86 is the sequence of the left primer for marker bnlg1755.

SEQ ID NO:87 is the sequence of the right primer for marker bnlg1755.

SEQ ID NO:88 is the sequence of the left primer for marker umc156a.

SEQ ID NO:89 is the sequence of the right primer for marker umc156a.

SEQ ID NO:90 is the sequence of the left primer for marker umc1142.

SEQ ID NO:91 is the sequence of the right primer for marker umc1142.

SEQ ID NO:92 is the sequence of the left primer for marker umc1346.

SEQ ID NO:93 is the sequence of the right primer for marker umc1346.

SEQ ID NO:94 is the sequence of the left primer for marker umc1702.

SEQ ID NO:95 is the sequence of the right primer for marker umc1702.

SEQ ID NO:96 is the sequence of the left primer for marker mmc0371.

SEQ ID NO:97 is the sequence of the right primer for marker mmc0371.

SEQ ID NO:98 is the sequence of the left primer for marker bnlg1621a.

SEQ ID NO:99 is the sequence of the right primer for marker bnlg1621a.

SEQ ID NO:100 is the sequence of the left primer for marker umc1299.

SEQ ID NO:101 is the sequence of the right primer for marker umc1299.

SEQ ID NO:102 is the sequence of the PHM 00045 reference sequence.

SEQ ID NO:103 is the sequence of the PHM 00049 reference sequence.

DETAILED DESCRIPTION OF THE INVENTION

The identification and selection of maize plants that show tolerance toGLS using MAS can provide an effective and environmentally friendlyapproach to overcoming losses caused by this disease. The presentinvention provides maize marker loci that demonstrate statisticallysignificant co-segregation with GLS tolerance. Detection of these locior additional linked loci can be used in marker assisted maize breedingprograms to produce tolerant plants, or plants with improved toleranceto GLS. The linked SSR and SNP markers identified herein are providedbelow and in the figures. These markers include PHM 15534, PHM 04694,PHM 01811, PHM 01963, PHM 05013, and PHM 00586 (FIG. 10).

Each SSR-type marker displays a plurality of alleles that can bevisualized as different sized PCR amplicons. The PCR primers that areused to generate the SSR-marker amplicons are provided in FIG. 9. Thealleles of SNP-type markers are determined using an allele-specifichybridization protocol, as known in the art. The PCR primers used toamplify the SNP domain are provided in FIG. 10.

As recognized in the art, any other marker that is linked to a QTLmarker (e.g., a disease tolerance marker) also finds use for that samepurpose. Examples of additional markers that are linked to the diseasetolerance markers recited herein are provided. For example, a linkedmarker can be determined from the closely linked markers provided inTable 3. It is not intended, however, that linked markers finding usewith the invention be limited to those recited in Table 3.

The invention also provides chromosomal QTL intervals that correlatewith GLS tolerance. These intervals are located on linkage group 4. Anymarker located within these intervals finds use as a marker for GLStolerance. These intervals include: (i) BNLG1755 and UMC1299, (ii)BNLG1755 and BNLG1621A, (iii) BNLG1755 and MMC0371, (iv) BNLG1755 andUMC1702, (v) UMC156A and UMC1299, (vi) UMC156A and BNLG1621A, (vii)UMC156A and MMC0371, (viii) UMC156A and UMC1702, (ix) UMC1142 andUMC1299, (x) UMC1142 and BNLG1621A, (xi) UMC1142 and MMC0371, (xii)UMC1142 and UMC1702, (xiii) UMC1346 and UMC1299, (xiv) UMC1346 andBNLG1621A, (xv) UMC1346 and MMC0371, and (xvi) UMC1346 and UMC1702.

The invention further provides a region of contiguous DNA bounded by andincluding PHM 00043 (SEQ ID NO:102) and PHM 00049 (SEQ ID NO:103), thathouses marker loci that cosegregate with GLS tolerance (FIG. 4). Anymarker locus lying within the contiguous span of DNA between andincluding SEQ ID NO:102, or a nucleotide sequence that is 95% identicalto SEQ ID NO:102 based on the Clustal V method of alignment, and SEQ IDNO:103, or a nucleotide sequence that is 95% identical to SEQ ID NO:103based on the Clustal V method of alignment, can find use as a marker forGLS tolerance.

Methods for identifying maize plants or germplasm that carry preferredalleles of tolerance marker loci are a feature of the invention. Inthese methods, any of a variety of marker detection protocols can beused to identify alleles at marker loci, depending on the type of markerlocus. Typical methods for detection include ASH, SSR detection, RFLPanalysis, and many others.

Although particular marker alleles can show co-segregation with adisease tolerance or susceptibility phenotype, it is important to notethat the marker locus is not necessarily part of the QTL locusresponsible for the tolerance or susceptibility. For example, it is nota requirement that the marker polynucleotide sequence be part of a genethat imparts disease tolerance (for example, be part of the gene openreading frame). The association between a specific marker allele withthe tolerance or susceptibility phenotype is due to the original“coupling” linkage phase between the marker allele and the QTL toleranceor susceptibility allele in the ancestral maize line from which thetolerance or susceptibility allele originated. Eventually, with repeatedrecombination, crossing over events between the marker and QTL locus canchange this orientation. For this reason, the favorable marker allelemay change depending on the linkage phase that exists within thetolerant parent used to create segregating populations. This does notchange the fact that the genetic marker can be used to monitorsegregation of the phenotype. It only changes which marker allele isconsidered favorable in a given segregating population.

Identification of maize plants or germplasm that contain marker allelesassociated with improved tolerance provides a basis for performingmarker assisted selection of maize. Maize plants that comprise favorablemarker alleles are selected for, while maize plants that comprise markeralleles that are negatively correlated with tolerance can be selectedagainst. Desired marker alleles can be introgressed into maize having adesired (e.g., elite or exotic) genetic background to produce anintrogressed tolerant maize plant or germplasm. In some aspects, it iscontemplated that a plurality of tolerance marker alleles aresequentially or simultaneous selected and/or introgressed. Thecombinations of tolerance markers that can be used to select fortolerance in a single plant are not limited, and can include anycombination of markers recited in FIGS. 3A and 3B, any markers linked tothe markers recited in FIGS. 3A and 3B, or any markers located withinthe QTL intervals defined herein.

As an alternative to standard breeding methods of introducing traits ofinterest into maize (e.g., introgression), transgenic approaches canalso be used. In these methods, exogenous nucleic acids controllingtraits of interest, e.g. disease tolerance, can be introduced intotarget plants or germplasm. Verification of tolerance can be performedby available tolerance protocols (as described, e.g., above). Toleranceassays are useful to verify that the tolerance trait still segregateswith the marker in any particular plant or population, and, of course,to measure the degree of tolerance improvement achieved by introgressingor transgenically introducing the trait into a desired background. Aplant comprising favorable alleles of a gray leaf spot QTL can have atolerance score of at least 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9,1.8, or 1.7 points greater, on a one to nine scale, when compared to anear isogenic plant not comprising the favorable alleles of the grayleaf spot QTL. In a 1 to 9 visual rating system, a higher scoreindicates a higher resistance. Data should be collected only whensufficient selection pressure exists in the experiment measured.

Systems, including automated systems for selecting plants that comprisea marker of interest and/or for correlating presence of the marker withtolerance are also a feature of the invention. These systems can includeprobes relevant to marker locus detection, detectors for detectinglabels on the probes, appropriate fluid handling elements andtemperature controllers that mix probes and templates and/or amplifytemplates, and systems instructions that correlate label detection tothe presence of a particular marker locus or allele.

Kits are also a feature of the invention. For example, a kit can includeappropriate primers or probes for detecting tolerance-associated markerloci and instructions in using the primers or probes for detecting themarker loci and correlating the loci with predicted GLS tolerance. Thekits can further include packaging materials for packaging the probes,primers or instructions, controls such as control amplificationreactions that include probes, primers or template nucleic acids foramplifications, molecular size markers, or the like.

Tolerance Markers and Favorable Alleles

In traditional linkage analysis, no direct knowledge of the physicalrelationship of genes on a chromosome is required. Mendel's first law isthat factors of pairs of characters are segregated, meaning that allelesof a diploid trait separate into two gametes and then into differentoffspring. Classical linkage analysis can be thought of as a statisticaldescription of the relative frequencies of cosegregation of differenttraits. Linkage analysis is the well characterized descriptive frameworkof how traits are grouped together based upon the frequency with whichthey segregate together. That is, if two non-allelic traits areinherited together with a greater than random frequency, they are saidto be “linked”. The frequency with which the traits are inheritedtogether is the primary measure of how tightly the traits are linked,i.e., traits which are inherited together with a higher frequency aremore closely linked than traits which are inherited together with lower(but still above random) frequency. Traits are linked because the geneswhich underlie the traits reside on the same chromosome. The furtherapart on a chromosome the genes reside, the less likely they are tosegregate together, because homologous chromosomes recombine duringmeiosis. Thus, the further apart on a chromosome the genes reside, themore likely it is that there will be a crossing over event duringmeiosis that will result in two genes segregating separately intoprogeny.

A common measure of linkage is the frequency with which traitscosegregate. This can be expressed as a percentage of cosegregation(recombination frequency) or, also commonly, in centiMorgans (cM). ThecM is named after the pioneering geneticist Thomas Hunt Morgan and is aunit of measure of genetic recombination frequency. One cM is equal to a1% chance that a trait at one genetic locus will be separated from atrait at another locus due to crossing over in a single generation(meaning the traits segregate together 99% of the time). Becausechromosomal distance is approximately proportional to the frequency ofcrossing over events between traits, there is an approximate physicaldistance that correlates with recombination frequency.

Marker loci are themselves traits and can be assessed according tostandard linkage analysis by tracking the marker loci duringsegregation. Thus, in the context of the present invention, one cM isequal to a 1% chance that a marker locus will be separated from anotherlocus (which can be any other trait, e.g., another marker locus, oranother trait locus that encodes a QTL), due to crossing over in asingle generation. The markers herein, as seen in FIGS. 3A and 3B, e.g.,PHM 15534, PHM 04694, PHM 01811, PHM 01963, PHM 05013, and PHM 00586, aswell as any of the chromosome intervals (i) BNLG1755 and UMC1299, (ii)BNLG1755 and BNLG1621A, (iii) BNLG1755 and MMC0371, (iv) BNLG1755 andUMC1702, (v) UMC156A and UMC1299, (vi) UMC156A and BNLG1621A, (vii)UMC156A and MMC0371, (viii) UMC156A and UMC1702, (ix) UMC1142 andUMC1299, (x) UMC1142 and BNLG1621A, (xi) UMC1142 and MMC0371, (xii)UMC1142 and UMC1702, (xiii) UMC1346 and UMC1299, (xiv) UMC1346 andBNLG1621A, (xv) UMC1346 and MMC0371, and (xvi) UMC1346 and UMC1702, havebeen found to correlate with newly conferred tolerance, enhancedtolerance, or susceptibility to GLS in maize. This means that themarkers are sufficiently closely linked to a tolerance trait that theycan be used as predictors for the tolerance trait. This is extremelyuseful in the context of marker assisted selection (MAS), discussed inmore detail herein. In brief, maize plants or germplasm can be selectedfor marker alleles that positively correlate with tolerance, withoutactually raising maize and measuring for newly conferred tolerance orenhanced tolerance (or, contrarily, maize plants can be selected againstif they possess markers that negatively correlate with newly conferredtolerance or enhanced tolerance). MAS is a powerful shortcut toselecting for desired phenotypes and for introgressing desired traitsinto cultivars of maize (e.g., introgressing desired traits into elitelines). MAS is easily adapted to high throughput molecular analysismethods that can quickly screen large numbers of plant or germplasmgenetic material for the markers of interest and is much more costeffective than raising and observing plants for visible traits.

In some embodiments, the QTL markers are a subset of the markersprovided in FIGS. 3A and 3B, for example, markers designed tobacb.pk0333.o19 (e.g., PHM 00043), bacc.pk0267.m12.f, cl33021_(—)1,bacb.pk0241.h17.f, chp2.pk0007.d2, p0094.csstg88, bacb.pk0269.n19,bacb.pk0009.b21.f, bacb.pk0117.i09.f, bacc.pk0280.n12, bacb.pk0219.j20,bacc.pk0132.b16.f, bacb.pk0221.o22, bacb.pk0544.j18, bacb.pk0540.c18.f,and bacm.pk022.b8 (e.g., PHM 00049).

When referring to the relationship between two genetic elements, such asa genetic element contributing to tolerance and a closely linked marker,“coupling” phase linkage indicates the state where the “favorable”allele at the tolerance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest (e.g., aQTL for tolerance) is physically linked with an “unfavorable” allele atthe closely linked marker locus, and the two “favorable” alleles are notinherited together (i.e., the two loci are “out of phase” with eachother).

A favorable allele of a marker is that allele of the marker thatco-segregates with a desired phenotype (e.g., disease tolerance). Asused herein, a QTL marker has a minimum of one favorable allele,although it is possible that the marker might have two or more favorablealleles found in the population. Any favorable allele of that marker canbe used advantageously for the identification and construction oftolerant maize lines. Optionally, one, two, three or more favorableallele(s) of different markers are identified in, or introgressed into aplant, and can be selected for or against during MAS. Desirably, plantsor germplasm are identified that have at least one such favorable allelethat positively correlates with newly conferred or enhanced tolerance.

Alternatively, a marker allele that is associated with diseasesusceptibility also finds use with the invention, since that allele canbe used to identify and counter select disease-susceptible plants. Suchan allele can be used for exclusionary purposes during breeding toidentify plants or germplasm that have alleles that negatively correlatewith tolerance, to eliminate susceptible plants or germplasm fromsubsequent rounds of breeding.

In some embodiments of the invention, a plurality of marker alleles aresimultaneously selected for in a single plant or a population of plants.In these methods, plants are selected that contain favorable alleles fortolerance to GLS, or favorable alleles for tolerance to GLS areintrogressed into a desired maize germplasm. One of skill in the artrecognizes that the simultaneous selection of favorable alleles for GLStolerance in the same plant is likely to result in an additive (or evensynergistic) protective effect for the plant.

One of skill recognizes that, in some cases, the identification offavorable marker alleles is germplasm-specific. The determination ofwhich marker alleles correlate with tolerance (or susceptibility) isdetermined for the particular germplasm under study. One of skillrecognizes that methods for identifying the favorable alleles areroutine and well known in the art, and furthermore, that theidentification and use of such favorable alleles is well within thescope of the invention. Furthermore still, identification of favorablemarker alleles in maize populations other than the populations used ordescribed herein is well within the scope of the invention.

Amplification primers for amplifying SSR-type marker loci are a featureof the invention. Another feature of the invention is primers specificfor the amplification of SNP domains (SNP markers), and the probes thatare used to genotype the SNP sequences. FIGS. 9 and 10 provide specificprimers for marker locus amplification and probes for detectingamplified marker loci. However, one of skill will immediately recognizethat other sequences to either side of the given primers can be used inplace of the given primers, so long as the primers can amplify a regionthat includes the allele to be detected. Further, it will be appreciatedthat the precise probe to be used for detection can vary, e.g., anyprobe that can identify the region of a marker amplicon to be detectedcan be substituted for those examples provided herein. Further, theconfiguration of the amplification primers and detection probes can, ofcourse, vary. Thus, the invention is not limited to the primers andprobes specifically recited herein.

In some aspects, methods of the invention utilize an amplification stepto detect/genotype a marker locus. However, it will be appreciated thatamplification is not a requirement for marker detection—for example, onecan directly detect unamplified genomic DNA simply by performing aSouthern blot on a sample of genomic DNA. Procedures for performingSouthern blotting, amplification (PCR, LCR, or the like) and many othernucleic acid detection methods are well established and are taught,e.g., in Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 (“Sambrook”); Current Protocols in Molecular Biology, F. M. Ausubelet al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (supplementedthrough 2002) (“Ausubel”) and PCR Protocols A Guide to Methods andApplications (Innis et al. eds) Academic Press Inc. San Diego, Calif.(1990) (“Innis”). Additional details regarding detection of nucleicacids in plants can also be found, e.g., in Plant Molecular Biology(1993) Croy (ed.) BIOS Scientific Publishers, Inc. (“Croy”).

Separate detection probes can also be omitted in amplification/detectionmethods, e.g., by performing a real time amplification reaction thatdetects product formation by modification of the relevant amplificationprimer upon incorporation into a product, incorporation of labelednucleotides into an amplicon, or by monitoring changes in molecularrotation properties of amplicons as compared to unamplified precursors(e.g., by fluorescence polarization).

Typically, molecular markers are detected by any established methodavailable in the art, including, without limitation, allele specifichybridization (ASH) or other methods for detecting single nucleotidepolymorphisms (SNP), amplified fragment length polymorphism (AFLP)detection, amplified variable sequence detection, randomly amplifiedpolymorphic DNA (RAPD) detection, restriction fragment lengthpolymorphism (RFLP) detection, self-sustained sequence replicationdetection, simple sequence repeat (SSR) detection, single-strandconformation polymorphisms (SSCP) detection, cleaved amplifiedpolymorphic sequences (CAPS) detection, isozyme markers detection, orthe like. While the exemplary markers provided in the figures and tablesherein are either SSR or SNP (ASH) markers, any of the aforementionedmarker types can be employed in the context of the invention to identifychromosome segments encompassing genetic element that contribute tosuperior agronomic performance (e.g., newly conferred tolerance orenhanced tolerance).

QTL Chromosome Intervals

In some aspects, the invention provides QTL chromosome intervals, whereone or more QTL associated with GLS tolerance are contained in thoseintervals. A variety of methods well known in the art are available foridentifying chromosome intervals (described in detail in the Examplesbelow). The boundaries of such chromosome intervals are drawn toencompass markers that will be linked to one or more QTL. In otherwords, the chromosome interval is drawn such that any marker that lieswithin that interval (including the terminal markers that define theboundaries of the interval) can be used as a marker for diseasetolerance. Each interval comprises a GLS QTL, and furthermore, mayindeed comprise more than one QTL. Close proximity of multiple QTL inthe same interval may obfuscate the correlation of a particular markerwith a particular QTL, as one marker may demonstrate linkage to morethan one QTL. Conversely, e.g., if two markers in close proximity showco-segregation with the desired phenotypic trait, it is sometimesunclear if each of those markers identifies the same QTL or twodifferent QTL.

The present invention provides maize chromosome intervals, where themarkers within that interval demonstrate co-segregation with toleranceto GLS (see Table 1).

TABLE 1 Method(s) of Flanking Markers Identification BNLG1755 andLinkage to a UMC1299 preferred marker BNLG1755 and Linkage to aBNLG1621A preferred marker BNLG1755 and Linkage to a MMC0371 preferredmarker BNLG1755 and Linkage to a UMC1702 preferred marker UMC156A andLinkage to a UMC1299 preferred marker UMC156A and Linkage to a BNLG1621Apreferred marker UMC156A and Linkage to a MMC0371 preferred markerUMC156A and Linkage to a UMC1702 preferred marker UMC1142 and Linkage toa UMC1299 preferred marker UMC1142 and Linkage to a BNLG1621A preferredmarker UMC1142 and Linkage to a MMC0371 preferred marker UMC1142 andLinkage to a UMC1702 preferred marker UMC1346 and Linkage to a UMC1299preferred marker UMC1346 and Linkage to a BNLG1621A preferred markerUMC1346 and Linkage to a MMC0371 preferred marker UMC1346 and Linkage toa UMC1702 preferred marker

Each of the intervals described above contains a clustering of markersthat can co-segregate with GLS tolerance. This clustering of markersoccurs in relatively small domains on the chromsome and can be linked toone or more QTL in those chromosome regions. QTL intervals were drawn toencompass the markers that co-segregate with tolerance. The intervalsare defined by the markers on their termini, where the intervalencompasses all the markers that map within the interval as well as themarkers that define the termini.

In some cases, an interval may be defined by linkage to a preferredmarker. For example, an interval on chromosome 4 is defined by anymarker that is linked to the marker PHM 01811 within a certain distance,referencing any suitable genetic linkage map. For example, as usedherein, linkage is defined as any marker that is within 25 cM of PHM01811, as defined by the IBM2 2004 Neighbors map found on the MaizeGDBwebsite. This interval on chromosome 4 is further illustrated in Table3. These markers are shown in genetic order. Each of the markers listed,including the terminal markers BNLG1755 and UMC1299, are members of theinterval. The BNLG1755 and UMC1299 markers are known in the art.

As described above, an interval (e.g., a chromosome interval or a QTLinterval) need not depend on an absolute measure of interval size suchas a centimorgan value. An interval can be described by the terminalmarkers that define the endpoints of the interval, and typically theinterval will include the terminal markers that define the extent of theinterval. For example, the physical map in FIG. 4 depicts the physicalregion of the chromosome bounded by and including PHM 00045 and PHM00049. An interval can include any marker localizing within thatchromosome domain, whether those markers are currently known or unknown.The invention provides a variety of means for defining a chromosomeinterval, for example, in the lists of linked markers of Table 3, and inreferences cited herein.

Genetic Maps

As one of skill in the art will recognize, recombination frequencies(and as a result, genetic map positions) in any particular populationare not static. The genetic distances separating two markers (or amarker and a QTL) can vary depending on how the map positions aredetermined. Variables such as the parents selected, the mappingpopulations used, the software used in the marker mapping or QTLmapping, and the parameters input by the user of the mapping softwarecan contribute to the QTL/marker genetic map relationships. For example,cM distances can very greatly depending on the number of recombinationcycles used to create the mapping population (e.g., IBM2=eightrecombination cycles, while BC1 herein is one recombination cycle; thus,the IBM2 distances of 5 cM are quite different from BC1 distances of 5cM). However, it is not intended that the invention be limited to anyparticular mapping populations, use of any particular software, or anyparticular set of software parameters to determine linkage of aparticular marker or chromosome interval with the GLS tolerancephenotype. It is well within the ability of one of ordinary skill in theart to extrapolate the novel features described herein to any maize genepool or population of interest, using any particular software andsoftware parameters. Indeed, observations regarding tolerance markersand chromosome intervals in populations in additions to those describedherein are readily made using the teaching of the present disclosure.

Mapping Software

A variety of commercial software is available for genetic mapping andmarker association studies (e.g., QTL mapping). This software includesbut is not limited to software listed in Table 2.

TABLE 2 Software Description/References Windows Wang S., C. J. Basten,and Z.-B. Zeng (2007). QTL Windows QTL Cartographer 2.5. Department ofCartographer Statistics, North Carolina State University, Version 2.5Raleigh, NC. JoinMap ® VanOoijen, and Voorrips (2001) “JoinMap 3.0software for the calculation of genetic linkage maps”, Plant ResearchInternational, Wageningen, the Netherlands; and Stam “Construction ofintegrated genetic linkage maps by means of a new computer package:JoinMap”, The Plant Journal 3(5): 739-744 (1993) MapQTL ® J. W.vanOoijen, “Software for the mapping of quantitative trait loci inexperimental populations”, Kyazma B. V., Wageningen, NetherlandsMapManager Manly and Olson, “Overview of QTL mapping software QT andintroduction to Map Manager QT”, Mamm. Genome 10: 327-334 (1999)MapManager Manly, Cudmore and Meer, “MapManager QTX, QTX cross-platformsoftware for genetic mapping”, Mamm. Genome 12: 930-932 (2001)GeneFlow ® GENEFLOW, Inc. (Alexandria, VA) and QTLocate ™ TASSEL (TraitAnalysis by aSSociation, Evolution, and Linkage) by Edward Buckler, andinformation about the program can be found on the Buckler Lab web pageat the Institute for Genomic Diversity at Cornell University.Unified Genetic Maps

“Unified”, “consensus”, or “integrated” genetic maps have been createdthat incorporate mapping data from two or more sources, includingsources that used different mapping populations and different modes ofstatistical analysis. The merging of genetic map information increasesthe marker density on the map. These improved maps can be advantageouslyused in marker assisted selection and map-based cloning and provide animproved framework for positioning newly identified molecular markers.The improved maps also aid in the identification of QTL chromosomeintervals and clusters of advantageously-linked markers.

In some aspects, a consensus map is derived by simply overlaying one mapon top of another. In other aspects, various algorithms, e.g., JoinMap®analysis, allows the combination of genetic mapping data from multiplesources, and reconciles discrepancies between mapping data from theoriginal sources. See, Van Ooijen and Voorrips (2001) “JoinMap 3.0software for the calculation of genetic linkage maps”, Plant ResearchInternational, Wageningen, the Netherlands; Stam (1993) “Construction ofintegrated genetic linkage maps by means of a new computer package:JoinMap”, The Plant Journal 3(5):739-744.

Linked Markers

From the present disclosure and widely recognized in the art, it isclear that any genetic marker that has a significant probability ofco-segregation with a phenotypic trait of interest (e.g., in the presentcase, a newly conferred tolerance or enhanced tolerance trait) can beused as a marker for that trait. A list of useful QTL markers providedby the present invention is provided in FIGS. 3A and 3B.

In addition to the QTL markers noted in FIGS. 3A and 3B, additionalmarkers linked to (showing linkage disequilibrium with) the QTL markerscan also be used to predict the newly conferred tolerance or enhancedtolerance trait in a maize plant. In other words, any other markershowing less than 50% recombination frequency (separated by a geneticdistance less than 50 cM) with a QTL marker of the invention (e.g., themarkers provided in FIGS. 3A and 3B) is also a feature of the invention.Any marker that is linked to a QTL marker can also be usedadvantageously in marker-assisted selection for the particular trait.

Genetic markers that are linked to QTL (e.g., QTL markers provided inFIGS. 3A and 3B) are particularly useful when they are sufficientlyclosely linked to a given QTL that they display a low recombinationfrequency. In the present invention, such closely linked markers are afeature of the invention. As defined herein, closely linked markersdisplay a recombination frequency of about 10% or less (the given markeris within 10 cM of the QTL). Put another way, these closely linked locico-segregate at least 90% of the time. Indeed, the closer a marker is toa QTL marker, the more effective and advantageous that marker becomes asan indicator for the desired trait.

Thus, in other embodiments, closely linked loci such as a QTL markerlocus and a second locus display an inter-locus cross-over frequency ofabout 10% or less, preferably about 9% or less, still more preferablyabout 8% or less, yet more preferably about 7% or less, still morepreferably about 6% or less, yet more preferably about 5% or less, stillmore preferably about 4% or less, yet more preferably about 3% or less,and still more preferably about 2% or less. In highly preferredembodiments, the relevant loci (e.g., a marker locus and a target locussuch as a QTL) display a recombination a frequency of about 1% or less,e.g., about 0.75% or less, more preferably about 0.5% or less, or yetmore preferably about 0.25% or less. Thus, the loci are about 10 cM, 9cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or0.25 cM or less apart. Put another way, two loci that are localized tothe same chromosome, and at such a distance that recombination betweenthe two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to beclosely linked to each other.

In some aspects, linked markers (including closely linked markers) ofthe invention are determined by review of a genetic map, for example,the integrated genetic maps found on the MaizeGDB website. For example,it is shown herein that the linkage group 4 markers PHM 15534, PHM04694, PHM 01811, PHM 01963, PHM 05013, and PHM 00586; and markersdesigned to bacb.pk0333.o19 (PHM 00043), bacc.pk0267.m12.f,cl33021_(—)1, bacb.pk0241.h17.f, chp2.pk0007.d2, p0094.csstg88,bacb.pk0269.n19, bacb.pk0009.b21.f, bacb.pk0117.i09.f, bacc.pk0280.n12,bacb.pk0219.j20, bacc.pk0132.b16.f, bacb.pk0221.o22, bacb.pk0544.j18,bacb.pk0540.c18.f, and bacm.pk022.b8 (PHM 00049) correlate with at leastone GLS tolerance QTL. Markers that are linked to the aforementionedmarkers can be determined, for example, from Table 3.

TABLE 3 Markers within 25 cM of PHM 01811 on IBM2 2004 Neighbors map MapPosition (IBM2 2004 Neighbors from Marker MaizeGDB website) BNLG1755299.90 UAZ73 300.09 BNL35B(BLR) 300.09 CSU81B(ANK) 300.11 MPIK19B 300.18UCSD72G 300.19 MMP45 300.20 MPIK11D 300.21 IAS12 300.36 NPI259A 300.38UCSD64F 300.38 UMC23B 300.46 BNLG1930 300.46 UMC156A 301.16 UAZ170301.16 UMC1142 302.50 NPI340B 304.16 UMC1346 304.30 UMC1702 305.20MMP155 305.50 UAZ47A 305.95 MMP149A 306.40 UMC1299 306.42

Similarly, linked markers (including closely linked markers) of theinvention can be determined by review of any suitable maize genetic map.For example, integrated genetic maps can be found on the MaizeGDBwebsite resource.

It is not intended that the determination of linked or closely linkedmarkers be limited to the use of any particular maize genetic map.Indeed, a large number of maize genetic maps is available and are wellknown to one of skill in the art. Alternatively, the determination oflinked and closely linked markers can be made by the generation of anexperimental dataset and linkage analysis.

It is also not intended that the identification of markers that arelinked (e.g., within about 50 cM or within about 10 cM) to the GLStolerance QTL markers identified herein be limited to any particular mapor methodology. The integrated genetic maps provided on the MaizeGDBwebsite serve only as example for identifying linked markers. Indeed,linked markers as defined herein can be determined from any genetic mapknown in the art (an experimental map or an integrated map), oralternatively, can be determined from any new mapping dataset.

It is noted that lists of linked and closely linked markers may varybetween maps and methodologies due to various factors. First, themarkers that are placed on any two maps may not be identical, andfurthermore, some maps may have a greater marker density than anothermap. Also, the mapping populations, methodologies and algorithms used toconstruct genetic maps can differ. One of skill in the art recognizesthat one genetic map is not necessarily more or less accurate thananother, and furthermore, recognizes that any maize genetic map can beused to determine markers that are linked and closely linked to the QTLmarkers of the present invention.

Techniques for Marker Detection

The invention provides molecular markers that have a significantprobability of co-segregation with QTL that impart a GLS tolerancephenotype. These QTL markers find use in marker assisted selection fordesired traits (newly conferred tolerance or enhanced tolerance), andalso have other uses. It is not intended that the invention be limitedto any particular method for the detection of these markers.

Markers corresponding to genetic polymorphisms between members of apopulation can be detected by numerous methods well-established in theart (e.g., PCR-based sequence specific amplification, restrictionfragment length polymorphisms (RFLPs), isozyme markers, allele specifichybridization (ASH), amplified variable sequences of the plant genome,self-sustained sequence replication, simple sequence repeat (SSR),single nucleotide polymorphism (SNP), random amplified polymorphic DNA(“RAPD”), cleaved amplified polymorphic sequences (CAPS), or amplifiedfragment length polymorphisms (AFLP)). In one additional embodiment, thepresence or absence of a molecular marker is determined simply throughnucleotide sequencing of the polymorphic marker region. This method isreadily adapted to high throughput analysis as are the other methodsnoted above, e.g., using available high throughput sequencing methodssuch as sequencing by hybridization.

In general, the majority of genetic markers rely on one or moreproperties of nucleic acids for their detection. For example, sometechniques for detecting genetic markers utilize hybridization of aprobe nucleic acid to nucleic acids corresponding to the genetic marker(e.g., amplified nucleic acids produced using genomic maize DNA as atemplate). Hybridization formats, including but not limited to solutionphase, solid phase, mixed phase, or in situ hybridization assays, areuseful for allele detection. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes Elsevier, New York; as well as in Sambrook and Ausubel (herein);and Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods inEnzymology volume 152 Academic Press, Inc., San Diego, Calif.(“Berger”).

For example, markers that comprise restriction fragment lengthpolymorphisms (RFLP) are detected, e.g., by hybridizing a probe which istypically a sub-fragment (or a synthetic oligonucleotide correspondingto a sub-fragment) of the nucleic acid to be detected to restrictiondigested genomic DNA. The restriction enzyme is selected to providerestriction fragments of at least two alternative (or polymorphic)lengths in different individuals or populations. Determining one or morerestriction enzymes that produce informative fragments for each cross isa simple procedure, well known in the art. After separation by length inan appropriate matrix (e.g., agarose or polyacrylamide) and transfer toa membrane (e.g., nitrocellulose, nylon, etc.), the labeled probe ishybridized under conditions which result in equilibrium binding of theprobe to the target followed by removal of excess probe by washing.

Nucleic acid probes to the marker loci can be cloned and/or synthesized.Any suitable label can be used with a probe of the invention. Detectablelabels suitable for use with nucleic acid probes include, for example,any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels include biotin for staining with labeledstreptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels,enzymes, and colorimetric labels. Other labels include ligands whichbind to antibodies labeled with fluorophores, chemiluminescent agents,and enzymes. A probe can also constitute radiolabelled PCR primers thatare used to generate a radiolabelled amplicon. Labeling strategies forlabeling nucleic acids and corresponding detection strategies can befound, e.g., in Haugland (1996) Handbook of Fluorescent Probes andResearch Chemicals Sixth Edition by Molecular Probes, Inc. (EugeneOreg.); or Haugland (2001) Handbook of Fluorescent Probes and ResearchChemicals Eighth Edition by Molecular Probes, Inc. (Eugene Oreg.)(Available on CD ROM).

Amplification-Based Detection Methods

PCR, RT-PCR and LCR are in particularly broad use as amplification andamplification-detection methods for amplifying nucleic acids of interest(e.g., those comprising marker loci), facilitating detection of themarkers. Details regarding the use of these and other amplificationmethods can be found in any of a variety of standard texts, including,e.g., Sambrook, Ausubel, Berger and Croy, herein. Many available biologytexts also have extended discussions regarding PCR and relatedamplification methods. One of skill will appreciate that essentially anyRNA can be converted into a double stranded DNA suitable for restrictiondigestion, PCR expansion and sequencing using reverse transcriptase anda polymerase (“Reverse Transcription-PCR, or “RT-PCR”). See also,Ausubel, Sambrook and Berger, above.

Real Time Amplification/Detection Methods

In one aspect, real time PCR or LCR is performed on the amplificationmixtures described herein, e.g., using molecular beacons or TaqMan™probes. A molecular beacon (MB) is an oligonucleotide or PNA which,under appropriate hybridization conditions, self-hybridizes to form astem and loop structure. The MB has a label and a quencher at thetermini of the oligonucleotide or PNA; thus, under conditions thatpermit intra-molecular hybridization, the label is typically quenched(or at least altered in its fluorescence) by the quencher. Underconditions where the MB does not display intra-molecular hybridization(e.g., when bound to a target nucleic acid, e.g., to a region of anamplicon during amplification), the MB label is unquenched. Detailsregarding standard methods of making and using MBs are well establishedin the literature, and MBs are available from a number of commercialreagent sources. See also, e.g., Leone et al. (1995) “Molecular beaconprobes combined with amplification by NASBA enable homogenous real-timedetection of RNA”, Nucleic Acids Res. 26:2150-2155; Tyagi and Kramer(1996) “Molecular beacons: probes that fluoresce upon hybridization”Nature Biotechnology 14:303-308; Blok and Kramer (1997) “Amplifiablehybridization probes containing a molecular switch” Mol Cell Probes11:187-194; Hsuih et al. (1997) “Novel, ligation-dependent PCR assay fordetection of hepatitis C in serum” J Clin Microbiol 34:501-507;Kostrikis et al. (1998) “Molecular beacons: spectral genotyping of humanalleles” Science 279:1228-1229; Sokol et al. (1998) “Real time detectionof DNA:RNA hybridization in living cells” Proc. Natl. Acad. Sci. U.S.A.95:11538-11543; Tyagi et al. (1998) “Multicolor molecular beacons forallele discrimination” Nature Biotechnology 16:49-53; Bonnet et al.(1999) “Thermodynamic basis of the chemical specificity of structuredDNA probes” Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176; Fang et al.(1999) “Designing a novel molecular beacon for surface-immobilized DNAhybridization studies” J. Am. Chem. Soc. 121:2921-2922; Marras et al.(1999) “Multiplex detection of single-nucleotide variation usingmolecular beacons” Genet. Anal. Biomol. Eng. 14:151-156; and Vet et al.(1999) “Multiplex detection of four pathogenic retroviruses usingmolecular beacons” Proc. Natl. Acad. Sci. U.S.A. 96:6394-6399.Additional details regarding MB construction and use is found in thepatent literature, e.g., U.S. Pat. Nos. 5,925,517, 6,150,097, and6,037,130.

PCR detection and quantification using dual-labeled fluorogenicoligonucleotide probes, commonly referred to as “TaqMan™ ” probes, canalso be performed according to the present invention. These probes arecomposed of short (e.g., 20-25 base) oligodeoxynucleotides that arelabeled with two different fluorescent dyes. On the 5′ terminus of eachprobe is a reporter dye, and on the 3′ terminus of each probe aquenching dye is found. The oligonucleotide probe sequence iscomplementary to an internal target sequence present in a PCR amplicon.When the probe is intact, energy transfer occurs between the twofluorophores and emission from the reporter is quenched by the quencherby FRET. During the extension phase of PCR, the probe is cleaved by 5′nuclease activity of the polymerase used in the reaction, therebyreleasing the reporter from the oligonucleotide-quencher and producingan increase in reporter emission intensity. Accordingly, TaqMan™ probesare oligonucleotides that have a label and a quencher, where the labelis released during amplification by the exonuclease action of thepolymerase used in amplification. This provides a real time measure ofamplification during synthesis. A variety of TaqMan™ reagents arecommercially available, e.g., from Applied Biosystems (DivisionHeadquarters in Foster City, Calif.) as well as from a variety ofspecialty vendors such as Biosearch Technologies (e.g., black holequencher probes).

Additional Details Regarding Amplified Variable Sequences, SSR, AFLP,ASH, SNPs and Isozyme Markers

Amplified variable sequences refer to amplified sequences of the plantgenome which exhibit high nucleic acid residue variability betweenmembers of the same species. All organisms have variable genomicsequences and each organism (with the exception of a clone) has adifferent set of variable sequences. Once identified, the presence ofspecific variable sequence can be used to predict phenotypic traits.Preferably, DNA from the plant serves as a template for amplificationwith primers that flank a variable sequence of DNA. The variablesequence is amplified and then sequenced.

Alternatively, self-sustained sequence replication can be used toidentify genetic markers. Self-sustained sequence replication refers toa method of nucleic acid amplification using target nucleic acidsequences which are replicated exponentially in vitro undersubstantially isothermal conditions by using three enzymatic activitiesinvolved in retroviral replication: (1) reverse transcriptase, (2) RNaseH, and (3) a DNA-dependent RNA polymerase (Guatelli et al. (1990) ProcNatl Acad Sci USA 87:1874). By mimicking the retroviral strategy of RNAreplication by means of cDNA intermediates, this reaction accumulatescDNA and RNA copies of the original target.

Amplified fragment length polymorphisms (AFLP) can also be used asgenetic markers (Vos et al. (1995) Nucleic Acids Res 23:4407). Thephrase “amplified fragment length polymorphism” refers to selectedrestriction fragments which are amplified before or after cleavage by arestriction endonuclease. The amplification step allows easier detectionof specific restriction fragments. AFLP allows the detection of largenumbers of polymorphic markers and has been used for genetic mapping ofplants (Becker et al. (1995) Mol Gen Genet 249:65; and Meksem et al.(1995) Mol Gen Genet 249:74).

Allele-specific hybridization (ASH) can be used to identify the geneticmarkers of the invention. ASH technology is based on the stableannealing of a short, single-stranded, oligonucleotide probe to acompletely complementary single-strand target nucleic acid. Detection isvia an isotopic or non-isotopic label attached to the probe.

For each polymorphism, two or more different ASH probes are designed tohave identical DNA sequences except at the polymorphic nucleotides. Eachprobe will have exact homology with one allele sequence so that therange of probes can distinguish all the known alternative allelesequences. Each probe is hybridized to the target DNA. With appropriateprobe design and hybridization conditions, a single-base mismatchbetween the probe and target DNA will prevent hybridization. In thismanner, only one of the alternative probes will hybridize to a targetsample that is homozygous or homogenous for an allele. Samples that areheterozygous or heterogeneous for two alleles will hybridize to both oftwo alternative probes.

ASH markers are used as dominant markers where the presence or absenceof only one allele is determined from hybridization or lack ofhybridization by only one probe. The alternative allele may be inferredfrom the lack of hybridization. ASH probe and target molecules areoptionally RNA or DNA; the target molecules are any length ofnucleotides beyond the sequence that is complementary to the probe; theprobe is designed to hybridize with either strand of a DNA target; theprobe ranges in size to conform to variously stringent hybridizationconditions, etc.

PCR allows the target sequence for ASH to be amplified from lowconcentrations of nucleic acid in relatively small volumes. Otherwise,the target sequence from genomic DNA is digested with a restrictionendonuclease and size separated by gel electrophoresis. Hybridizationstypically occur with the target sequence bound to the surface of amembrane or, as described in U.S. Pat. No. 5,468,613, the ASH probesequence may be bound to a membrane.

In one embodiment, ASH data are typically obtained by amplifying nucleicacid fragments (amplicons) from genomic DNA using PCR, transferring theamplicon target DNA to a membrane in a dot-blot format, hybridizing alabeled oligonucleotide probe to the amplicon target, and observing thehybridization dots by autoradiography.

Single nucleotide polymorphisms (SNP) are markers that consist of ashared sequence differentiated on the basis of a single nucleotide. Thisdistinction can be detected by differential migration patterns of anamplicon comprising the SNP on, e.g., an acrylamide gel. Alternativemodes of detection, such as hybridization, e.g., ASH, or RFLP analysisare also appropriate. SNP markers detect single base pair nucleotidesubstitutions. Of all the molecular marker types, SNPs are the mostabundant, thus having the potential to provide the highest genetic mapresolution (Bhattramakki et al. 2002 Plant Molecular Biology48:539-547). SNPs can be assayed at an even higher level of throughputthan SSRs, in a so-called ‘ultra-high-throughput’ fashion, as they donot require large amounts of DNA and automation of the assay may bestraight-forward. SNPs also have the promise of being relativelylow-cost systems. These three factors together make SNPs highlyattractive for use in MAS. Several methods are available for SNPgenotyping, including but not limited to, hybridization, primerextension, oligonucleotide ligation, nuclease cleavage, minisequencingand coded spheres. Such methods have been reviewed in: Gut (2001) HumMutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000)Pharmacogenomics 1, pp. 95-100; Bhattramakki and Rafalski (2001)Discovery and application of single nucleotide polymorphism markers inplants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting ofPlants, CABI Publishing, Wallingford. A wide range of commerciallyavailable technologies utilize these and other methods to interrogateSNPs including Masscode™ (Qiagen), Invader® (Third Wave Technologies),SnapShot® (Applied Biosystems), Taqman® (Applied Biosystems) andBeadarrays™ (Illumina).

A number of SNPs together within a sequence, or across linked sequences,can be used to describe a haplotype for any particular genotype (Chinget al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001, Rafalski (2002b),supra). Haplotypes can be more informative than single SNPs and can bemore descriptive of any particular genotype. For example, a single SNPmay be allele ‘T’ for PHM 01811-32, but the allele ‘T’ might also occurin the maize breeding population being utilized for recurrent parents.In this case, a haplotype, e.g. a series of alleles at linked SNPmarkers, may be more informative. Once a unique haplotype has beenassigned to a donor chromosomal region, that haplotype can be used inthat population or any subset thereof to determine whether an individualhas a particular gene. See, for example, WO2003054229. Using automatedhigh throughput marker detection platforms known to those of ordinaryskill in the art makes this process highly efficient and effective.

Isozyme markers can be employed as genetic markers, e.g., to trackmarkers other than the tolerance markers herein, or to track isozymemarkers linked to the markers herein. Isozymes are multiple forms ofenzymes that differ from one another in their amino acid sequences, andtherefore their nucleic acid sequences. Some isozymes are multimericenzymes containing slightly different subunits. Other isozymes areeither multimeric or monomeric but have been cleaved from the proenzymeat different sites in the amino acid sequence. Isozymes can becharacterized and analyzed at the protein level, or alternatively,isozymes which differ at the nucleic acid level can be determined. Insuch cases any of the nucleic acid based methods described herein can beused to analyze isozyme markers.

Additional Details Regarding Nucleic Acid Amplification

As noted, nucleic acid amplification techniques such as PCR and LCR arewell known in the art and can be applied to the present invention toamplify and/or detect nucleic acids of interest, such as nucleic acidscomprising marker loci. Examples of techniques sufficient to directpersons of skill through such in vitro methods, including the polymerasechain reaction (PCR), the ligase chain reaction (LCR), Qββ-replicaseamplification and other RNA polymerase mediated techniques (e.g.,NASBA), are found in the references noted above, e.g., Innis, Sambrook,Ausubel, Berger and Croy. Additional details are found in Mullis et al.(1987) U.S. Pat. No. 4,683,202; Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3:81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87:1874; Lomeli et al. (1989) J. Clin. Chem 35:1826;Landegren et al. (1988) Science 241:1077-1080; Van Brunt (1990)Biotechnology 8:291-294; Wu and Wallace (1989) Gene 4: 560; Barringer etal. (1990) Gene 89:117; and Sooknanan and Malek (1995) Biotechnology13:563-564. Improved methods of amplifying large nucleic acids by PCR,which is useful in the context of positional cloning, are furthersummarized in Cheng et al. (1994) Nature 369:684, and the referencestherein, in which PCR amplicons of up to 40 kb are generated.

Detection of Markers for Positional Cloning

In some embodiments, a nucleic acid probe is used to detect a nucleicacid that comprises a marker sequence. Such probes can be used, forexample, in positional cloning to isolate nucleotide sequences linked tothe marker nucleotide sequence. It is not intended that the nucleic acidprobes of the invention be limited to any particular size. In someembodiments, a nucleic acid probe is at least 20 nucleotides in length,or alternatively, at least 50 nucleotides in length, or alternatively,at least 100 nucleotides in length, or alternatively, at least 200nucleotides in length.

A hybridized probe is detected using autoradiography, fluorography orother similar detection techniques depending on the label to bedetected. Examples of specific hybridization protocols are widelyavailable in the art, see, e.g., Berger, Sambrook, and Ausubel, allherein.

Probe/Primer Synthesis Methods

In general, synthetic methods for making oligonucleotides, includingprobes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids),etc., are well known. For example, oligonucleotides can be synthesizedchemically according to the solid phase phosphoramidite triester methoddescribed by Beaucage and Caruthers (1981), Tetrahedron Letts.22(20):1859-1862, e.g., using a commercially available automatedsynthesizer, e.g., as described in Needham-VanDevanter et al. (1984)Nucleic Acids Res. 12:6159-6168. Oligonucleotides, including modifiedoligonucleotides, can also be ordered from a variety of commercialsources known to persons of skill. There are many commercial providersof oligo synthesis services, and thus, this is a broadly accessibletechnology. Any nucleic acid can be custom ordered from any of a varietyof commercial sources, such as The Midland Certified Reagent Company,The Great American Gene Company, ExpressGen Inc., Operon TechnologiesInc. (Alameda, Calif.), and many others. Similarly, PNAs can be customordered from any of a variety of sources, such as PeptidoGenic, HTIBio-Products, Inc., BMA Biomedicals Ltd (U.K.), Bio•Synthesis, Inc., andmany others.

In Silico Marker Detection

In alternative embodiments, in silico methods can be used to detect themarker loci of interest. For example, the sequence of a nucleic acidcomprising the marker locus of interest can be stored in a computer. Thedesired marker locus sequence or its homolog can be identified using anappropriate nucleic acid search algorithm as provided by, for example,in such readily available programs as BLAST, or even simple wordprocessors.

Amplification Primers for Marker Detection

In some preferred embodiments, the molecular markers of the inventionare detected using a suitable PCR-based detection method, where the sizeor sequence of the PCR amplicon is indicative of the absence or presenceof a particular marker allele. In these types of methods, PCR primersare hybridized to the conserved regions flanking the polymorphic markerregion. As used in the art, PCR primers used to amplify a molecularmarker are sometimes termed “PCR markers” or simply “markers”.

It will be appreciated that, although many specific examples of primersare provided herein (see, e.g., FIGS. 3A and 3B, FIG. 9, and FIG. 10),suitable primers to be used with the invention can be designed using anysuitable method. It is not intended that the invention be limited to anyparticular primer or primer pair. For example, primers can be designedusing any suitable software program, such as LASERGENE®.

In some embodiments, the primers of the invention are radiolabelled, orlabeled by any suitable means (e.g., using a non-radioactive fluorescenttag), to allow for rapid visualization of the different size ampliconsfollowing an amplification reaction and size separation, e.g. byelectrophoresis on agarose gel. In some embodiments, the primers are notlabeled, and the amplicons are visualized following their sizeresolution with, e.g., ethidium bromide staining.

It is not intended that the primers of the invention be limited togenerating an amplicon of any particular size. For example, the primersused to amplify the marker loci and alleles herein are not limited toamplifying the entire region of the relevant locus. The primers cangenerate an amplicon of any suitable length. In some embodiments, markeramplification produces an amplicon at least 20 nucleotides in length, oralternatively, at least 50 nucleotides in length, or alternatively, atleast 100 nucleotides in length, or alternatively, at least 200nucleotides in length.

Marker Assisted Selection and Breeding of Plants

A primary motivation for development of molecular markers in cropspecies is the potential for increased efficiency in plant breedingthrough marker assisted selection (MAS). Genetic markers are used toidentify plants that contain a desired genotype at one or more loci, andthat are expected to transfer the desired genotype, along with a desiredphenotype, to their progeny. Genetic markers can be used to identifyplants that contain a desired genotype at one locus, or at severalunlinked or linked loci (e.g., a haplotype), and that would be expectedto transfer the desired genotype, along with a desired phenotype totheir progeny. The present invention provides the means to identifyplants, particularly maize plants, that have newly conferred toleranceor enhanced tolerance to, or are susceptible to, GLS by identifyingplants having a specified allele at one of those loci, e.g., PHM 15534,PHM 04694, PHM 01811, PHM 01963, PHM 05013, and PHM 00586; and markersdesigned to bacb.pk0333.o19 (PHM 00043), bacc.pk0267.m12.f,cl33021_(—)1, bacb.pk0241.h17.f, chp2.pk0007.d2, p0094.csstg88,bacb.pk0269.n19, bacb.pk0009.b21.f, bacb.pk0117.i09.f, bacc.pk0280.n12,bacb.pk0219.j20, bacc.pk0132.b16.f, bacb.pk0221.o22, bacb.pk0544.j18,bacb.pk0540.c18.f, and bacm.pk022.b8 (PHM 00049).

Similarly, by identifying plants lacking the desired marker locus,susceptible or less tolerant plants can be identified and, e.g.,eliminated from subsequent crosses. Similarly, these marker loci can beintrogressed into any desired genomic background, germplasm, plant,line, variety, etc., as part of an overall MAS breeding program designedto enhance maize yield.

The invention also provides chromosome QTL intervals that find equal usein MAS to select plants that demonstrate newly conferred or enhanced GLStolerance. Similarly, the QTL intervals can also be used tocounter-select plants that are susceptible or have reduced tolerance toGLS. Any marker that maps within the QTL interval (including the terminiof the intervals) finds use with the invention. These intervals aredefined by the following pairs of markers: (i) BNLG1755 and UMC1299,(ii) BNLG1755 and BNLG1621A, (iii) BNLG1755 and MMC0371, (iv) BNLG1755and UMC1702, (v) UMC156A and UMC1299, (vi) UMC156A and BNLG1621A, (vii)UMC156A and MMC0371, (viii) UMC156A and UMC1702, (ix) UMC1142 andUMC1299, (x) UMC1142 and BNLG1621A, (xi) UMC1142 and MMC0371, (xii)UMC1142 and UMC1702, (xiii) UMC1346 and UMC1299, (xiv) UMC1346 andBNLG1621A, (xv) UMC1346 and MMC0371, and (xvi) UMC1346 and UMC1702.

In general, MAS uses polymorphic markers that have been identified ashaving a significant likelihood of co-segregation with a tolerancetrait. Such markers are presumed to map near a gene or genes that givethe plant its tolerance phenotype, and are considered indicators for thedesired trait, and are termed QTL markers. Plants are tested for thepresence of a desired allele at the QTL marker. The most preferredmarkers (or marker alleles) are those that have the strongestassociation with the tolerance trait.

Linkage analysis is used to determine which polymorphic marker alleledemonstrates a statistical likelihood of co-segregation with thetolerance phenotype (thus, a “tolerance marker allele”). Following theidentification of a marker allele for co-segregation with the tolerancephenotype, it is possible to use this marker for rapid, accuratescreening of plant lines for the tolerance allele without the need togrow the plants through their life cycle and await phenotypicevaluations, and furthermore, permits genetic selection for theparticular tolerance allele even when the molecular identity of theactual tolerance QTL is unknown. Tissue samples can be taken, forexample, from the first leaf of the plant and screened with theappropriate molecular marker, and it is rapidly determined which progenywill advance. Linked markers also remove the impact of environmentalfactors that can often influence phenotypic expression.

A polymorphic QTL marker locus can be used to select plants that containthe marker allele (or alleles) that correlate with the desired tolerancephenotype. In brief, a nucleic acid corresponding to the marker nucleicacid allele is detected in a biological sample from a plant to beselected. This detection can take the form of hybridization of a probenucleic acid to a marker allele or amplicon thereof, e.g., usingallele-specific hybridization, Southern analysis, northern analysis, insitu hybridization, hybridization of primers followed by PCRamplification of a region of the marker, or the like. A variety ofprocedures for detecting markers are described herein, e.g., in thesection entitled “TECHNIQUES FOR MARKER DETECTION”. After the presence(or absence) of a particular marker allele in the biological sample isverified, the plant is selected (e.g., used to make progeny plants byselective breeding).

Maize plant breeders desire combinations of tolerance loci with genesfor high yield and other desirable traits to develop improved maizevarieties. Screening large numbers of samples by non-molecular methods(e.g., trait evaluation in maize plants) can be expensive, timeconsuming, and unreliable. Use of the polymorphic markers describedherein, when genetically-linked to tolerance loci, provide an effectivemethod for selecting tolerant varieties in breeding programs. Forexample, one advantage of marker-assisted selection over fieldevaluations for tolerance is that MAS can be done at any time of year,regardless of the growing season. Moreover, environmental effects arelargely irrelevant to marker-assisted selection.

When a population is segregating for multiple loci affecting one ormultiple traits, e.g., multiple loci involved in tolerance, or multipleloci each involved in tolerance to different diseases, the efficiency ofMAS compared to phenotypic screening becomes even greater, because allthe loci can be evaluated in the lab together from a single sample ofDNA. In the present instance, the PHM 15534, PHM 04694, PHM 01811, PHM01963, PHM 05013, and PHM 00586; and markers designed to bacb.pk0333.o19(e.g., PHM 00043), bacc.pk0267.m12.f, cl33021_(—)1, bacb.pk0241.h17.f,chp2.pk0007.d2, p0094.csstg88, bacb.pk0269.n19, bacb.pk0009.b21.f,bacb.pk0117.i09.f, bacc.pk0280.n12, bacb.pk0219.j20, bacc.pk0132.b16.f,bacb.pk0221.o22, bacb.pk0544.j18, bacb.pk0540.c18.f, and bacm.pk022.b8(e.g., PHM 00049) markers, as well as any of the chromosome intervals(i) BNLG1755 and UMC1299, (ii) BNLG1755 and BNLG1621A, (iii) BNLG1755and MMC0371, (iv) BNLG1755 and UMC1702, (v) UMC156A and UMC1299, (vi)UMC156A and BNLG1621A, (vii) UMC156A and MMC0371, (viii) UMC156A andUMC1702, (ix) UMC1142 and UMC1299, (x) UMC1142 and BNLG1621A, (xi)UMC1142 and MMC0371, (xii) UMC1142 and UMC1702, (xiii) UMC1346 andUMC1299, (xiv) UMC1346 and BNLG1621A, (xv) UMC1346 and MMC0371, and(xvi) UMC1346 and UMC1702, can be assayed simultaneously or sequentiallyfrom a single sample or a population of samples.

Another use of MAS in plant breeding is to assist the recovery of therecurrent parent genotype by backcross breeding. Backcross breeding isthe process of crossing a progeny back to one of its parents or parentlines. Backcrossing is usually done for the purpose of introgressing oneor a few loci from a donor parent (e.g., a parent comprising desirabletolerance marker loci) into an otherwise desirable genetic backgroundfrom the recurrent parent (e.g., an otherwise high yielding maize line).The more cycles of backcrossing that are done, the greater the geneticcontribution of the recurrent parent to the resulting introgressedvariety. This is often necessary, because tolerant plants may beotherwise undesirable, e.g., due to low yield, low fecundity, or thelike. In contrast, strains which are the result of intensive breedingprograms may have excellent yield, fecundity or the like, merely beingdeficient in one desired trait such as tolerance to GLS.

The presence and/or absence of a particular genetic marker or allele,e.g., PHM 15534, PHM 04694, PHM 01811, PHM 01963, PHM 05013, and PHM00586; and markers designed to bacb.pk0333.o19 (e.g., PHM 00043),bacc.pk0267.m12.f, cl33021_(—)1, bacb.pk0241.h17.f, chp2.pk0007.d2,p0094.csstg88, bacb.pk0269.n19, bacb.pk0009.b21.f, bacb.pk0117.i09.f,bacc.pk0280.n12, bacb.pk0219.j20, bacc.pk0132.b16.f, bacb.pk0221.o22,bacb.pk0544.j18, bacb.pk0540.c18.f, and bacm.pk022.b8 (e.g., PHM 00049)markers, as well as any of the chromosome intervals (i) BNLG1755 andUMC1299, (ii) BNLG1755 and BNLG1621A, (iii) BNLG1755 and MMC0371, (iv)BNLG1755 and UMC1702, (v) UMC156A and UMC1299, (vi) UMC156A andBNLG1621A, (vii) UMC156A and MMC0371, (viii) UMC156A and UMC1702, (ix)UMC1142 and UMC1299, (x) UMC1142 and BNLG1621A, (xi) UMC1142 andMMC0371, (xii) UMC1142 and UMC1702, (xiii) UMC1346 and UMC1299, (xiv)UMC1346 and BNLG1621A, (xv) UMC1346 and MMC0371, and (xvi) UMC1346 andUMC1702, in the genome of a plant is made by any method noted herein. Ifthe nucleic acids from the plant are positive for a desired geneticmarker allele, the plant can be self fertilized to create a truebreeding line with the same genotype, or it can be crossed with a plantwith the same marker or with other desired characteristics to create asexually crossed hybrid generation.

Introgression of Favorable Alleles—Efficient Backcrossing of ToleranceMarkers into Elite Lines

One application of MAS, in the context of the present invention, is touse the newly conferred tolerance or enhanced tolerance markers toincrease the efficiency of an introgression or backcrossing effort aimedat introducing a tolerance QTL into a desired (typically high yielding)background. In marker assisted backcrossing of specific markers (andassociated QTL) from a donor source, e.g., to an elite or exotic geneticbackground, one selects among backcross progeny for the donor trait andthen uses repeated backcrossing to the elite or exotic line toreconstitute as much of the elite/exotic background's genome aspossible.

Thus, the markers and methods of the present invention can be utilizedto guide marker assisted selection or breeding of maize varieties withthe desired complement (set) of allelic forms of chromosome segmentsassociated with superior agronomic performance (tolerance, along withany other available markers for yield, etc.). Any of the disclosedmarker alleles can be introduced into a maize line via introgression, bytraditional breeding (or introduced via transformation, or both), toyield a maize plant with superior agronomic performance. The number ofalleles associated with tolerance that can be introduced or be presentin a maize plant of the present invention ranges from one to the numberof alleles disclosed herein, each integer of which is incorporatedherein as if explicitly recited.

The present invention also extends to a method of making a progeny maizeplant and these progeny maize plants, per se. The method comprisescrossing a first parent maize plant with a second maize plant andgrowing the female maize plant under plant growth conditions to yieldmaize plant progeny. Methods of crossing and growing maize plants arewell within the ability of those of ordinary skill in the art. Suchmaize plant progeny can be assayed for alleles associated with toleranceand, thereby, the desired progeny selected. Such progeny plants or seedcan be sold commercially for maize production, used for food, processedto obtain a desired constituent of the maize, or further utilized insubsequent rounds of breeding. At least one of the first or second maizeplants is a maize plant of the present invention in that it comprises atleast one of the allelic forms of the markers of the present invention,such that the progeny are capable of inheriting the allele.

A method of the present invention can be applied to at least one relatedmaize plant such as from progenitor or descendant lines in the subjectmaize plant's pedigree such that inheritance of the desired toleranceallele can be traced. The number of generations separating the maizeplants being subject to the methods of the present invention willgenerally be from 1 to 20, commonly 1 to 5, and typically 1, 2, or 3generations of separation, and quite often a direct descendant or parentof the maize plant will be subject to the method (i.e., one generationof separation).

Introgression of Favorable Alleles—Incorporation of “Exotic” Germplasmwhile Maintaining Breeding Progress

Genetic diversity is important for long term genetic gain in anybreeding program. With limited diversity, genetic gain will eventuallyplateau when all the favorable alleles have been fixed within the elitepopulation. One objective is to incorporate diversity into an elite poolwithout losing the genetic gain that has already been made and with theminimum possible investment. MAS provide an indication of which genomicregions and which favorable alleles from the original ancestors havebeen selected for and conserved over time, facilitating efforts toincorporate favorable variation from exotic germplasm sources (parentsthat are unrelated to the elite gene pool) in the hopes of findingfavorable alleles that do not currently exist in the elite gene pool.

For example, the markers of the present invention can be used for MAS incrosses involving elite x exotic maize lines by subjecting thesegregating progeny to MAS to maintain major yield alleles, along withthe tolerance marker alleles herein.

Positional Cloning

The molecular marker loci and alleles of the present invention, e.g.,PHM 15534, PHM 04694, PHM 01811, PHM 01963, PHM 05013, and PHM 00586;and markers designed to bacb.pk0333.o19 (e.g., PHM 00043),bacc.pk0267.m12.f, cl33021_(—)1, bacb.pk0241.h17.f, chp2.pk0007.d2,p0094.csstg88, bacb.pk0269.n19, bacb.pk0009.b21.f, bacb.pk0117.i09.f,bacc.pk0280.n12, bacb.pk0219.j20, bacc.pk0132.b16.f, bacb.pk0221.o22,bacb.pk0544.j18, bacb.pk0540.c18.f, and bacm.pk022.b8 (e.g., PHM 00049)markers, as well as any of the chromosome intervals (i) BNLG1755 andUMC1299, (ii) BNLG1755 and BNLG1621A, (iii) BNLG1755 and MMC0371, (iv)BNLG1755 and UMC1702, (v) UMC156A and UMC1299, (vi) UMC156A andBNLG1621A, (vii) UMC156A and MMC0371, (viii) UMC156A and UMC1702, (ix)UMC1142 and UMC1299, (x) UMC1142 and BNLG1621A, (xi) UMC1142 andMMC0371, (xii) UMC1142 and UMC1702, (xiii) UMC1346 and UMC1299, (xiv)UMC1346 and BNLG1621A, (xv) UMC1346 and MMC0371, and (xvi) UMC1346 andUMC1702, can be used, as indicated previously, to identify a toleranceQTL, which can be cloned by well established procedures, e.g., asdescribed in detail in Ausubel, Berger and Sambrook, herein.

These tolerance clones are first identified by their genetic linkage tomarkers of the present invention. Isolation of a nucleic acid ofinterest is achieved by any number of methods as discussed in detail insuch references as Ausubel, Berger and Sambrook, herein, and Clark, Ed.(1997) Plant Molecular Biology: A Laboratory Manual Springer-Verlag,Berlin.

For example, “positional gene cloning” uses the proximity of a tolerancemarker to physically define an isolated chromosomal fragment containinga tolerance QTL gene. The isolated chromosomal fragment can be producedby such well known methods as digesting chromosomal DNA with one or morerestriction enzymes, or by amplifying a chromosomal region in apolymerase chain reaction (PCR), or any suitable alternativeamplification reaction. The digested or amplified fragment is typicallyligated into a vector suitable for replication and, e.g., expression, ofthe inserted fragment. Markers that are adjacent to an open readingframe (ORF) associated with a phenotypic trait can hybridize to a DNAclone (e.g., a clone from a genomic DNA library), thereby identifying aclone on which an ORF (or a fragment of an ORF) is located. If themarker is more distant, a fragment containing the ORF is identified bysuccessive rounds of screening and isolation of clones which togethercomprise a contiguous sequence of DNA, a process termed “chromosomewalking”, resulting in a “contig” or “contig map”. Protocols sufficientto guide one of skill through the isolation of clones associated withlinked markers are found in, e.g., Berger, Sambrook and Ausubel, allherein.

Generation of Transgenic Cells and Plants

The present invention also relates to host cells and organisms which aretransformed with nucleic acids corresponding to tolerance QTL identifiedaccording to the invention. For example, such nucleic acids includechromosome intervals (e.g., genomic fragments), ORFS and/or cDNAs thatencode a newly conferred tolerance or enhanced tolerance trait.Additionally, the invention provides for the production of polypeptidesthat provide newly conferred tolerance or enhanced tolerance byrecombinant techniques.

General texts which describe molecular biological techniques for thecloning and manipulation of nucleic acids and production of encodedpolypeptides include Berger, Sambrook, and Ausubel supra. These textsdescribe mutagenesis, the use of vectors, promoters and many otherrelevant topics related to, e.g., the generation of clones that comprisenucleic acids of interest, e.g., marker loci, marker probes, QTL thatsegregate with marker loci, etc.

Host cells are genetically engineered (e.g., transduced, transfected,transformed, etc.) with the vectors of this invention (e.g., vectors,such as expression vectors which comprise an ORF derived from or relatedto a tolerance QTL) which can be, for example, a cloning vector, ashuttle vector or an expression vector. Such vectors are, for example,in the form of a plasmid, a phagemid, an agrobacterium, a virus, a nakedpolynucleotide (linear or circular), or a conjugated polynucleotide.Vectors can be introduced into bacteria, especially for the purpose ofpropagation and expansion. The vectors are also introduced into planttissues, cultured plant cells or plant protoplasts by a variety ofstandard methods known in the art, including but not limited toelectroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82:5824),infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohnet al. (1982) Molecular Biology of Plant Tumors (Academic Press, NewYork), pp. 549-560; U.S. Pat. No. 4,407,956), high velocity ballisticpenetration by small particles with the nucleic acid either within thematrix of small beads or particles, or on the surface (Klein et al.(1987) Nature 327:70), use of pollen as vector (WO85/01856), or use ofAgrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid inwhich DNA fragments are cloned. The T-DNA plasmid is transmitted toplant cells upon infection by Agrobacterium tumefaciens, and a portionis stably integrated into the plant genome (Horsch et al. (1984) Science233:496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803).Additional details regarding nucleic acid introduction methods are foundin Sambrook, Berger and Ausubel, supra. The method of introducing anucleic acid of the present invention into a host cell is not criticalto the instant invention, and it is not intended that the invention belimited to any particular method for introducing exogenous geneticmaterial into a host cell. Thus, any suitable method, e.g., includingbut not limited to the methods provided herein, which provides foreffective introduction of a nucleic acid into a cell or protoplast canbe employed and finds use with the invention.

The engineered host cells can be cultured in conventional nutrient mediamodified as appropriate for such activities as, for example, activatingpromoters or selecting transformants. These cells can optionally becultured into transgenic plants. In addition to Sambrook, Berger andAusubel, supra, plant regeneration from cultured protoplasts isdescribed in Evans et al. (1983) “Protoplast Isolation and Culture”,Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co.),New York; Davey (1983) “Recent Developments in the Culture andRegeneration of Plant Protoplasts”, Protoplasts, pp. 12-29, (Birkhauser,Basel); Dale (1983) “Protoplast Culture and Plant Regeneration ofCereals and Other Recalcitrant Crops”, Protoplasts pp. 31-41,(Birkhauser, Basel); Binding (1985) “Regeneration of Plants”, PlantProtoplasts, pp. 21-73, (CRC Press, Boca Raton, Fla.). Additionaldetails regarding plant cell culture and regeneration include Payne etal. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley &Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell,Tissue and Organ Culture; Fundamental Methods Springer Lab Manual,Springer-Verlag (Berlin Heidelberg New York) and Plant Molecular Biology(1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN0 12 198370 6. Cell culture media in general are also set forth in Atlasand Parks (eds) The Handbook of Microbiological Media (1993) CRC Press,Boca Raton, Fla. Additional information for cell culture is found inavailable commercial literature such as the Life Science Research CellCulture Catalogue (1998) from Sigma-Aldrich, Inc. (St Louis, Mo.)(“Sigma-LSRCCC”) and, e.g., the Plant Culture Catalogue and supplement(e.g., 1997 or later) also from Sigma-Aldrich, Inc. (St Louis, Mo.)(“Sigma-PCCS”).

The present invention also relates to the production of transgenicorganisms, which may be bacteria, yeast, fungi, animals or plants,transduced with the nucleic acids of the invention (e.g., nucleic acidscomprising the marker loci and/or QTL noted herein). A thoroughdiscussion of techniques relevant to bacteria, unicellular eukaryotesand cell culture is found in references enumerated herein and arebriefly outlined as follows. Several well-known methods of introducingtarget nucleic acids into bacterial cells are available, any of whichmay be used in the present invention. These include: fusion of therecipient cells with bacterial protoplasts containing the DNA, treatmentof the cells with liposomes containing the DNA, electroporation,projectile bombardment (biolistics), carbon fiber delivery, andinfection with viral vectors (discussed further, below), etc. Bacterialcells can be used to amplify the number of plasmids containing DNAconstructs of this invention. The bacteria are grown to log phase, andthe plasmids within the bacteria can be isolated by a variety of methodsknown in the art (see, for instance, Sambrook). In addition, a plethoraof kits are commercially available for the purification of plasmids frombacteria. For their proper use, follow the manufacturer's instructions(see, for example, EasyPrep™, FlexiPrep™, both from Pharmacia Biotech;StrataClean™, from Stratagene; and, QIAprep™ from Qiagen). The isolatedand purified plasmids are then further manipulated to produce otherplasmids, used to transfect plant cells or incorporated intoAgrobacterium tumefaciens related vectors to infect plants. Typicalvectors contain transcription and translation terminators, transcriptionand translation initiation sequences, and promoters useful forregulation of the expression of the particular target nucleic acid. Thevectors optionally comprise generic expression cassettes containing atleast one independent terminator sequence, sequences permittingreplication of the cassette in eukaryotes, or prokaryotes, or both,(e.g., shuttle vectors) and selection markers for both prokaryotic andeukaryotic systems. Vectors are suitable for replication and integrationin prokaryotes, eukaryotes, or preferably both. See, Giliman & Smith(1979) Gene 8:81; Roberts et al. (1987) Nature 328:731; Schneider et al.(1995) Protein Expr. Purif. 6:10; Ausubel, Sambrook, Berger (all supra).A catalogue of Bacteria and Bacteriophages useful for cloning isprovided, e.g., by the American Type Culture Collection (ATCC), e.g.,The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gherna et al.(eds) published by the ATCC. Additional basic procedures for sequencing,cloning and other aspects of molecular biology and underlyingtheoretical considerations are also found in Watson et al. (1992)Recombinant DNA, Second Edition, Scientific American Books, NY. Inaddition, essentially any nucleic acid (and virtually any labelednucleic acid, whether standard or non-standard) can be custom orstandard ordered from any of a variety of commercial sources, such asthe Midland Certified Reagent Company (Midland, Tex.), The GreatAmerican Gene Company (Ramona, Calif.), ExpressGen Inc. (Chicago, Ill.),Operon Technologies Inc. (Alameda, Calif.) and many others.

Introducing Nucleic Acids into Plants.

Embodiments of the present invention pertain to the production oftransgenic plants comprising the cloned nucleic acids, e.g., isolatedORFS and cDNAs encoding tolerance genes. Techniques for transformingplant cells with nucleic acids are widely available and can be readilyadapted to the invention. In addition to Berger, Ausubel and Sambrook,all supra, useful general references for plant cell cloning, culture andregeneration include Jones (ed) (1995) Plant Gene Transfer andExpression Protocols—Methods in Molecular Biology, Volume 49 HumanaPress Towata N.J. (“Jones”); Payne et al. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.(“Payne”); and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue andOrgan Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag(Berlin Heidelberg New York) (“Gamborg”). A variety of cell culturemedia are described in Atlas and Parks (eds) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, Fla. (“Atlas”).Additional information for plant cell culture is found in availablecommercial literature such as the Life Science Research Cell CultureCatalogue (1998) from Sigma-Aldrich, Inc. (St Louis, Mo.) (Sigma-LSRCCC)and, e.g., the Plant Culture Catalogue and supplement (1997) also fromSigma-Aldrich, Inc. (St Louis, Mo.) (Sigma-PCCS). Additional detailsregarding plant cell culture are found in Croy.

The nucleic acid constructs of the invention, e.g., plasmids, cosmids,artificial chromosomes, DNA and RNA polynucleotides, are introduced intoplant cells, either in culture or in the organs of a plant by a varietyof conventional techniques. Where the sequence is expressed, thesequence is optionally combined with transcriptional and translationalinitiation regulatory sequences which direct the transcription ortranslation of the sequence from the exogenous DNA in the intendedtissues of the transformed plant.

Isolated nucleic acid acids of the present invention can be introducedinto plants according to any of a variety of techniques known in theart. Techniques for transforming a wide variety of higher plant speciesare also well known and described in widely available technical,scientific, and patent literature. See, for example, Weising et al.(1988) Ann. Rev. Genet. 22:421-477.

The DNA constructs of the invention, for example plasmids, phagemids,cosmids, phage, naked or variously conjugated-DNA polynucleotides (e.g.,polylysine-conjugated DNA, peptide-conjugated DNA, liposome-conjugatedDNA), or artificial chromosomes, can be introduced directly into thegenomic DNA of the plant cell using techniques such as electroporationand microinjection of plant cell protoplasts, or the DNA constructs canbe introduced directly to plant cells using ballistic methods, such asDNA particle bombardment.

Microinjection techniques for injecting plant, e.g., cells, embryos,callus and protoplasts, are known in the art and well described in thescientific and patent literature. For example, a number of methods aredescribed in Jones, as well as in the other references noted herein andavailable in the literature.

For example, the introduction of DNA constructs using polyethyleneglycol precipitation is described in Paszkowski et al., EMBO J. 3:2717(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniquesare described in Klein et al., Nature 327:70-73 (1987). Additionaldetails are found in Jones and Gamborg, supra, and in U.S. Pat. No.5,990,387.

Alternatively, and in some cases preferably, Agrobacterium-mediatedtransformation is employed to generate transgenic plants.Agrobacterium-mediated transformation techniques, including disarmingand use of binary vectors, are also well described in the scientificliterature. See, for example, Horsch, et al. (1984) Science 233:496;Fraley et al. (1984) Proc. Natl. Acad. Sci. USA 80:4803: and reviewed inHansen and Chilton (1998) Curr. Top. Microbiol. Immunol. 240:21 and Das(1998) Subcellular Biochemistry 29: Plant Microbe Interactions, pp.343-363.

DNA constructs are optionally combined with suitable T-DNA flankingregions and introduced into a conventional Agrobacterium tumefacienshost vector. The virulence functions of the Agrobacterium tumefacienshost will direct the insertion of the construct and adjacent marker intothe plant cell DNA when the cell is infected by the bacteria. See, U.S.Pat. No. 5,591,616. Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, PWJRigby, Ed., London, Academic Press; and Lichtenstein and Draper (1985)In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press; WO88/02405, published Apr. 7, 1988, describes the use of A. rhizogenesstrain Δ4 and its Ri plasmid along with A. tumefaciens vectors pARC8 orpARC16), (2) liposome-mediated DNA uptake (see, e.g., Freeman et al.(1984) Plant Cell Physiol. 25:1353), and (3) the vortexing method (see,e.g., Kindle (1990) Proc. Natl. Acad. Sci. (USA) 87:1228).

DNA can also be introduced into plants by direct DNA transfer intopollen as described by Zhou et al. (1983) Meth. Enzymol. 101:433; D.Hess (1987) Intern Rev. Cytol. 107:367; Luo et al. (1988) Plant Mol.Biol. Rep. 6:165. Expression of polypeptide coding genes can be obtainedby injection of the DNA into reproductive organs of a plant as describedby Pena et al. (1987) Nature 325:274. DNA can also be injected directlyinto the cells of immature embryos and the desiccated embryos rehydratedas described by Neuhaus et al. (1987) Theor. Appl. Genet. 75:30; andBenbrook et al. (1986) in Proceedings Bio Expo Butterworth, Stoneham,Mass., pp. 27-54. A variety of plant viruses that can be employed asvectors are known in the art and include cauliflower mosaic virus(CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.

Generation/Regeneration of Transgenic Plants

Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantthat possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Payne; Fundamental Methods Springer LabManual, Springer-Verlag (Berlin Heidelberg New York); Evans et al.(1983) Protoplasts Isolation and Culture, Handbook of Plant Cell Culturepp. 124-176, Macmillian Publishing Company, New York; and Binding (1985)Regeneration of Plants, Plant Protoplasts pp. 21-73, CRC Press, BocaRaton. Regeneration can also be obtained from plant callus, explants,somatic embryos (Dandekar et al. (1989) J. Tissue Cult. Meth. 12:145;McGranahan et al. (1990) Plant Cell Rep. 8:512) organs, or partsthereof. Such regeneration techniques are described generally in Klee etal. (1987) Ann. Rev. Plant Phys. 38:467-486. Additional details arefound in Payne and Jones, both supra, and Weissbach and Weissbach, eds.(1988) Methods for Plant Molecular Biology Academic Press, Inc., SanDiego, Calif. This regeneration and growth process includes the steps ofselection of transformant cells and shoots, rooting the transformantshoots, and growth of the plantlets in soil. These methods are adaptedto the invention to produce transgenic plants bearing QTL and othergenes isolated according to the methods of the invention.

In addition, the regeneration of plants containing polynucleotides ofthe present invention and introduced by Agrobacterium into cells of leafexplants can be achieved as described by Horsch et al. (1985) Science227:1229-1231. In this procedure, transformants are grown in thepresence of a selection agent and in a medium that induces theregeneration of shoots in the plant species being transformed asdescribed by Fraley et al. (1983) Proc. Natl. Acad. Sci. (U.S.A.)80:4803. This procedure typically produces shoots within two to fourweeks, and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

It is not intended that plant transformation and expression ofpolypeptides that provide disease tolerance, as provided by the presentinvention, be limited to maize species. Indeed, it is contemplated thatthe polypeptides that provide the desired tolerance in maize can alsoprovide such tolerance when transformed and expressed in otheragronomically and horticulturally important species. For example, suchspecies include: soybean, canola, alfalfa, wheat, sunflower, andsorghum.

In construction of recombinant expression cassettes of the invention,which include, for example, helper plasmids comprising virulencefunctions, and plasmids or viruses comprising exogenous DNA sequencessuch as structural genes, a plant promoter fragment is optionallyemployed which directs expression of a nucleic acid in any or alltissues of a regenerated plant. Examples of constitutive promotersinclude the cauliflower mosaic virus (CaMV) 35S transcription initiationregion, the 1′- or 2′-promoter derived from T-DNA of Agrobacteriumtumefaciens, and other transcription initiation regions from variousplant genes known to those of skill. Alternatively, the plant promotermay direct expression of the polynucleotide of the invention in aspecific tissue (tissue-specific promoters) or may be otherwise undermore precise environmental control (inducible promoters). Examples oftissue-specific promoters under developmental control include promotersthat initiate transcription only in certain tissues, such as fruit,seeds or flowers.

Any of a number of promoters which direct transcription in plant cellscan be suitable. The promoter can be either constitutive or inducible.In addition to the promoters noted above, promoters of bacterial originthat operate in plants include the octopine synthase promoter, thenopaline synthase promoter and other promoters derived from native Tiplasmids. See, Herrara-Estrella et al. (1983) Nature 303:209. Viralpromoters include the 35S and 19S RNA promoters of cauliflower mosaicvirus. See, Odell et al. (1985) Nature 313:810. Other plant promotersinclude Kunitz trypsin inhibitor promoter (KTI), SCP1, SUP, UCD3, theribulose-1,3-bisphosphate carboxylase small subunit promoter and thephaseolin promoter. The promoter sequence from the E8 gene and othergenes may also be used. The isolation and sequence of the E8 promoter isdescribed in detail in Deikman and Fischer (1988) EMBO J. 7:3315. Manyother promoters are in current use and can be coupled to an exogenousDNA sequence to direct expression of the nucleic acid.

If expression of a polypeptide from a cDNA is desired, a polyadenylationregion at the 3′-end of the coding region is typically included. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant genes, or from, e.g., T-DNA.

The vector comprising the sequences (e.g., promoters or coding regions)from genes encoding expression products and transgenes of the inventionwill typically include a nucleic acid subsequence, a marker gene whichconfers a selectable, or alternatively, a screenable, phenotype on plantcells. For example, the marker can encode biocide tolerance,particularly antibiotic tolerance, such as tolerance to kanamycin, G418,bleomycin, hygromycin, or herbicide tolerance, such as tolerance tochlorosulforon, or phosphinothricin (the active ingredient in theherbicides bialaphos or Basta). See, e.g., Padgette et al. (1996) In:Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis Publishers,Boca Raton. For example, crop selectivity to specific herbicides can beconferred by engineering genes into crops that encode appropriateherbicide metabolizing enzymes from other organisms, such as microbes.See, Vasil (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91,CRC Lewis Publishers, Boca Raton).

One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. In vegetatively propagated crops, maturetransgenic plants can be propagated by the taking of cuttings or bytissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype. Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like, are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants, are also included within thescope of the invention, provided that these parts comprise theintroduced nucleic acid sequences.

Transgenic or introgressed plants expressing a polynucleotide of thepresent invention can be screened for transmission of the nucleic acidof the present invention by, for example, standard nucleic aciddetection methods or by immunoblot protocols. Expression at the RNAlevel can be determined to identify and quantitate expression-positiveplants. Standard techniques for RNA analysis can be employed and includeRT-PCR amplification assays using oligonucleotide primers designed toamplify only heterologous or introgressed RNA templates and solutionhybridization assays using marker or linked QTL specific probes. Plantscan also be analyzed for protein expression, e.g., by Western immunoblotanalysis using antibodies that recognize the encoded polypeptides. Inaddition, in situ hybridization and immunocytochemistry according tostandard protocols can be done using heterologous nucleic acid specificpolynucleotide probes and antibodies, respectively, to localize sites ofexpression within transgenic tissue. Generally, a number of transgeniclines are usually screened for the incorporated nucleic acid to identifyand select plants with the most appropriate expression profiles.

One embodiment of the invention is a transgenic plant that is homozygousfor the added heterologous nucleic acid; e.g., a transgenic plant thatcontains two added nucleic acid sequence copies, e.g., a gene at thesame locus on each chromosome of a homologous chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating(self-fertilizing) a heterozygous transgenic plant that contains asingle added heterologous nucleic acid, germinating some of the seedproduced and analyzing the resulting plants produced for alteredexpression of a polynucleotide of the present invention relative to acontrol plant (e.g., a native, non-transgenic plant). Back-crossing to aparental plant and out-crossing with a non-transgenic plant can be usedto introgress the heterologous nucleic acid into a selected background(e.g., an elite or exotic maize line).

Methods for Assessing and Producing GLS Tolerant Maize Plants

Experienced plant breeders can recognize tolerant maize plants in thefield and can select the tolerant individuals or populations forbreeding purposes or for propagation. In this context, the plant breederrecognizes “tolerant” and “non-tolerant”, or “susceptible”, maizeplants.

Such plant breeding practitioners will appreciate that plant toleranceis a phenotypic spectrum consisting of extremes in tolerance andsusceptibility as well as a continuum of intermediate tolerancephenotypes. Tolerance also varies due to environmental effects and theseverity of pathogen infection. Evaluation of phenotypes usingreproducible assays and tolerance scoring methods are of value toscientists who seek to identify genetic loci that impart tolerance,conduct marker assisted selection for tolerant populations, and breedtolerance traits into elite maize lines via introgression techniques,for example.

In contrast to fortuitous field observations that classify plants aseither “tolerant” or “susceptible”, various systems are known forscoring the degree of plant tolerance or susceptibility. Thesetechniques can be applied to different fields at different times, andprovide approximate tolerance scores that can be used to characterize agiven strain regardless of growth conditions or location.

This invention also is directed to methods for producing a maize plantby crossing a first parent maize plant with a second parent maize plantwherein either the first or second parent maize plant is an inbred maizeplant of the line PHJEP. Further, both first and second parent maizeplants can come from the inbred maize line PHJEP. Still further, thisinvention also is directed to methods for producing an inbred maize linePHJEP-derived maize plant by crossing inbred maize line PHJEP with asecond maize plant, growing the progeny seed, and repeating the crossingand growing steps with the inbred maize line PHJEP-derived plant 0 to 5times. Thus, any such methods using the inbred maize line PHJEP are partof this invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using inbred maize linePHJEP as a parent are within the scope of this invention, includingplants derived from inbred maize line PHJEP. Advantageously, the inbredmaize line is used in crosses with other, different, maize inbreds toproduce first generation (F1) maize hybrid seeds and plants withsuperior characteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which maize plants can be regenerated,plant calli, plant clumps, and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, kernels,ears, cobs, leaves, husks, stalks, roots, root tips, anthers, silk andthe like.

Duncan et al., Planta (1985) 165:322-332 reflects that 97% of the plantscultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad et al., in Plant Cell Reports (1988), 7:262-265 reports severalmedia additions that enhance regenerability of callus of two inbredlines. Other published reports also indicated that “nontraditional”tissues are capable of producing somatic embryogenesis and plantregeneration. K. P. Rao, et al., Maize Genetics Cooperation Newsletter,60:64-65 (1986), refers to somatic embryogenesis from glume calluscultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987)indicates somatic embryogenesis from the tissue cultures of maize leafsegments. Thus, it is clear from the literature that the state of theart is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of maize is described in European Patent Application,publication 160,390, incorporated herein by reference. Maize tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, Va. 1982, at367-372) and in Duncan, et al., “The Production of Callus Capable ofPlant Regeneration from Immature Embryos of Numerous Zea MaysGenotypes,” 165 Planta 322-332 (1985). Thus, another aspect of thisinvention is to provide cells which upon growth and differentiationproduce maize plants having the physiological and morphologicalcharacteristics of inbred line PHJEP.

The utility of inbred maize line PHJEP also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Coix,Schierachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with PHJEP may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

Automated Detection/Correlation Systems of the Invention

In some embodiments, the present invention includes an automated systemfor detecting markers of the invention and/or correlating the markerswith a desired phenotype (e.g., tolerance). Thus, a typical system caninclude a set of marker probes or primers configured to detect at leastone favorable allele of one or more marker locus associated with newlyconferred tolerance or enhanced tolerance to GLS. These probes orprimers are configured to detect the marker alleles noted in the tablesand examples herein, e.g., using any available allele detection format,e.g., solid or liquid phase array based detection, microfluidic-basedsample detection, etc.

For example, in one embodiment, the marker locus is PHM 15534, PHM04694, PHM 01811, PHM 01963, PHM 05013, and PHM 00586, or anycombination thereof, as well as any of the chromosome intervals (i)BNLG1755 and UMC1299, (ii) BNLG1755 and BNLG1621A, (iii) BNLG1755 andMMC0371, (iv) BNLG1755 and UMC1702, (v) UMC156A and UMC1299, (vi)UMC156A and BNLG1621A, (vii) UMC156A and MMC0371, (viii) UMC156A andUMC1702, (ix) UMC1142 and UMC1299, (x) UMC1142 and BNLG1621A, (xi)UMC1142 and MMC0371, (xii) UMC1142 and UMC1702, (xiii) UMC1346 andUMC1299, (xiv) UMC1346 and BNLG1621A, (xv) UMC1346 and MMC0371, and(xvi) UMC1346 and UMC1702, or any combination thereof, and the probe setis configured to detect the locus.

The typical system includes a detector that is configured to detect oneor more signal outputs from the set of marker probes or primers, oramplicon thereof, thereby identifying the presence or absence of theallele. A wide variety of signal detection apparatus are available,including photo multiplier tubes, spectrophotometers, CCD arrays, arraysand array scanners, scanning detectors, phototubes and photodiodes,microscope stations, galvo-scanns, microfluidic nucleic acidamplification detection appliances and the like. The preciseconfiguration of the detector will depend, in part, on the type of labelused to detect the marker allele, as well as the instrumentation that ismost conveniently obtained for the user. Detectors that detectfluorescence, phosphorescence, radioactivity, pH, charge, absorbance,luminescence, temperature, magnetism or the like can be used. Typicaldetector embodiments include light (e.g., fluorescence) detectors orradioactivity detectors. For example, detection of a light emission(e.g., a fluorescence emission) or other probe label is indicative ofthe presence or absence of a marker allele. Fluorescent detection isespecially preferred and is generally used for detection of amplifiednucleic acids (however, upstream and/or downstream operations can alsobe performed on amplicons, which can involve other detection methods).In general, the detector detects one or more label (e.g., light)emission from a probe label, which is indicative of the presence orabsence of a marker allele.

The detector(s) optionally monitors one or a plurality of signals froman amplification reaction. For example, the detector can monitor opticalsignals which correspond to “real time” amplification assay results.

System instructions that correlate the presence or absence of thefavorable allele with the predicted tolerance are also a feature of theinvention. For example, the instructions can include at least onelook-up table that includes a correlation between the presence orabsence of the favorable allele and the predicted newly conferredtolerance or enhanced tolerance. The precise form of the instructionscan vary depending on the components of the system, e.g., they can bepresent as system software in one or more integrated unit of the system(e.g., a microprocessor, computer or computer readable medium), or canbe present in one or more units (e.g., computers or computer readablemedia) operably coupled to the detector. As noted, in one typicalembodiment, the system instructions include at least one look-up tablethat includes a correlation between the presence or absence of thefavorable allele and predicted newly conferred tolerance or enhancedtolerance. The instructions also typically include instructionsproviding a user interface with the system, e.g., to permit a user toview results of a sample analysis and to input parameters into thesystem.

The system typically includes components for storing or transmittingcomputer readable data representing or designating the alleles detectedby the methods of the present invention, e.g., in an automated system.The computer readable media can include cache, main, and storage memoryand/or other electronic data storage components (hard drives, floppydrives, storage drives, etc.) for storage of computer code. Datarepresenting alleles detected by the method of the present invention canalso be electronically, optically, or magnetically transmitted in acomputer data signal embodied in a transmission medium over a networksuch as an intranet or internet or combinations thereof. The system canalso or alternatively transmit data via wireless, IR, or other availabletransmission alternatives.

During operation, the system typically comprises a sample that is to beanalyzed, such as a plant tissue, or material isolated from the tissuesuch as genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA,amplified RNA, or the like.

The phrase “allele detection/correlation system” in the context of thisinvention refers to a system in which data entering a computercorresponds to physical objects or processes external to the computer,e.g., a marker allele, and a process that, within a computer, causes aphysical transformation of the input signals to different outputsignals. In other words, the input data, e.g., amplification of aparticular marker allele is transformed to output data, e.g., theidentification of the allelic form of a chromosome segment. The processwithin the computer is a set of instructions, or “program”, by whichpositive amplification or hybridization signals are recognized by theintegrated system and attributed to individual samples as a genotype.Additional programs correlate the identity of individual samples withphenotypic values or marker alleles, e.g., statistical methods. Inaddition there are numerous C/C++ programs for computing, Delphi and/orJava programs for GUI interfaces, and productivity tools (e.g.,Microsoft Excel and/or SigmaPlot) for charting or creating look uptables of relevant allele-trait correlations. Other useful softwaretools in the context of the integrated systems of the invention includestatistical packages such as SAS, Genstat, Matlab, Mathematica, andS-Plus and genetic modeling packages such as QU-GENE. Furthermore,additional programming languages such as visual basic are also suitablyemployed in the integrated systems of the invention.

For example, tolerance marker allele values assigned to a population ofprogeny descending from crosses between elite lines are recorded in acomputer readable medium, thereby establishing a database correspondingtolerance alleles with unique identifiers for members of the populationof progeny. Any file or folder, whether custom-made or commerciallyavailable (e.g., from Oracle or Sybase), suitable for recording data ina computer readable medium is acceptable as a database in the context ofthe present invention. Data regarding genotype for one or more molecularmarkers, e.g., ASH, SSR, RFLP, RAPD, AFLP, SNP, CAPS, isozyme markers orother markers as described herein, are similarly recorded in a computeraccessible database. Optionally, marker data is obtained using anintegrated system that automates one or more aspects of the assay (orassays) used to determine marker(s) genotype. In such a system, inputdata corresponding to genotypes for molecular markers are relayed from adetector, e.g., an array, a scanner, a CCD, or other detection devicedirectly to files in a computer readable medium accessible to thecentral processing unit. A set of system instructions (typicallyembodied in one or more programs) encoding the correlations betweentolerance and the alleles of the invention is then executed by thecomputational device to identify correlations between marker alleles andpredicted trait phenotypes.

Typically, the system also includes a user input device, such as akeyboard, a mouse, a touchscreen, or the like (for, e.g., selectingfiles, retrieving data, reviewing tables of maker information), and anoutput device (e.g., a monitor, a printer) for viewing or recovering theproduct of the statistical analysis.

Thus, in one aspect, the invention provides an integrated systemcomprising a computer or computer readable medium comprising a set offiles and/or a database with at least one data set that corresponds tothe marker alleles herein. The system also includes a user interfaceallowing a user to selectively view one or more of these databases. Inaddition, standard text manipulation software such as word processingsoftware (e.g., Microsoft Word™ or Corel WordPerfect™) and database orspreadsheet software (e.g., spreadsheet software such as MicrosoftExcel™, Corel Quattro Pro™, or database programs such as MicrosoftAccess™ or Paradox™) can be used in conjunction with a user interface(e.g., a GUI in a standard operating system such as a Windows,Macintosh, Unix or Linux system) to manipulate strings of characterscorresponding to the alleles or other features of the database.

The systems optionally include components for sample manipulation, e.g.,incorporating robotic devices. For example, a robotic liquid controlarmature for transferring solutions (e.g., plant cell extracts) from asource to a destination, e.g., from a microtiter plate to an arraysubstrate, is optionally operably linked to the digital computer (or toan additional computer in the integrated system). An input device forentering data to the digital computer to control high throughput liquidtransfer by the robotic liquid control armature and, optionally, tocontrol transfer by the armature to the solid support is commonly afeature of the integrated system. Many such automated robotic fluidhandling systems are commercially available. For example, a variety ofautomated systems are available from Caliper Technologies (Hopkinton,Mass.), which utilize various Zymate systems, which typically include,e.g., robotics and fluid handling modules. Similarly, the common ORCA®robot, which is used in a variety of laboratory systems, e.g., formicrotiter tray manipulation, is also commercially available, e.g., fromBeckman Coulter, Inc. (Fullerton, Calif.). As an alternative toconventional robotics, microfluidic systems for performing fluidhandling and detection are now widely available, e.g., from CaliperTechnologies Corp. (Hopkinton, Mass.) and Agilent Technologies (PaloAlto, Calif.).

Systems for molecular marker analysis of the present invention can thusinclude a digital computer with one or more of high-throughput liquidcontrol software, image analysis software for analyzing data from markerlabels, data interpretation software, a robotic liquid control armaturefor transferring solutions from a source to a destination operablylinked to the digital computer, an input device (e.g., a computerkeyboard) for entering data to the digital computer to control highthroughput liquid transfer by the robotic liquid control armature and,optionally, an image scanner for digitizing label signals from labeledprobes hybridized, e.g., to markers on a solid support operably linkedto the digital computer. The image scanner interfaces with the imageanalysis software to provide a measurement of, e.g., nucleic acid probelabel intensity upon hybridization to an arrayed sample nucleic acidpopulation (e.g., comprising one or more markers), where the probe labelintensity measurement is interpreted by the data interpretation softwareto show whether, and to what degree, the labeled probe hybridizes to amarker nucleic acid (e.g., an amplified marker allele). The data soderived is then correlated with sample identity, to determine theidentity of a plant with a particular genotype(s) for particular markersor alleles, e.g., to facilitate marker assisted selection of maizeplants with favorable allelic forms of chromosome segments involved inagronomic performance (e.g., newly conferred tolerance or enhancedtolerance).

Optical images, e.g., hybridization patterns viewed (and, optionally,recorded) by a camera or other recording device (e.g., a photodiode anddata storage device) are optionally further processed in any of theembodiments herein, e.g., by digitizing the image and/or storing andanalyzing the image on a computer. A variety of commercially availableperipheral equipment and software is available for digitizing, storingand analyzing a digitized video or digitized optical image, e.g., usingPC (e.g., Intel x86 or Pentium chip-compatible DOS™, OS2™, WINDOWS™,WINDOWS NT™, WINDOWS 95™, WINDOWS 97™, WINDOWS 2000™, WINDOWS XP™, orWINDOWS VISTA™ based machines), MACINTOSH™, LINUX, or UNIX based (e.g.,SUN™ work station) computers.

EXAMPLES

The following examples are offered to illustrate, but not to limit, theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only, and persons skilledin the art will recognize various reagents or parameters that can bealtered without departing from the spirit of the invention or the scopeof the appended claims.

Example 1 Mapping a Large Effect QTL for GLS Tolerance

The GLS source of tolerance is uncommon in North American (NA)germplasm. To create an elite line for use as a donor source for GLStolerance, the GLS tolerant line PH14T (U.S. Pat. No. 5,942,670) wascrossed to PHN46 (U.S. Pat. No. 5,567,861) in order to generate an F1population. Individuals of the F1 population were later backcrossed toPHN46 to generate a BC5 backcross population of approximately 190individuals.

The F1 populations were phenotyped in the field for GLS tolerance.Marker data was collected on each progeny individual using SSR markers,with good genome coverage to generate a molecular marker map for thepopulation. QTL analysis of the data from the F1 population identified aQTL for GLS tolerance on Chromosome 4, bin 5, between markers UMC1791and UMC1346.

The BC5 population was also phenotyped in the field for GLS resistance.Marker data was collected on each progeny individual using SSR markers,with good genome coverage to generate a molecular marker map for thepopulation. The BC5 mapping population confirmed the location of the QTLfor GLS tolerance and more precisely identified the QTL for GLStolerance as being closely linked to BNLG1755. This QTL showed a largeand consistent effect, and explained 8-44% of the total phenotypicvariation (Table 4).

TABLE 4 Single marker regression QTL mapping of GLS tolerance from BC5Population (all markers on Chromosome 4) WN & Average Marker WN R2 D21CR2 WWDGTF R2 NH R2 GLS R2 PHI079 0.000 6.3 0.000 2.9 0.000 1.9 0.000 5.60.000 1.9 BNLG1937 0.000 13.7 0.000 8.4 0.000 1.1 0.000 9.5 0.000 1.1BNLG1265 0.000 20.1 0.000 11.1 0.000 6.7 0.000 17.9 0.000 6.7 BNLG17550.000 44.4 0.000 7.5 0.000 10.2 0.000 25.9 0.000 10.2 UMC1175 0.000 8.40.000 3.4 0.000 6.9 0.000 6.0 0.000 6.9 BNLG1189 0.000 6.0 0.015 0.60.019 1.8 0.000 4.4 0.000 1.8

Example 2 Fine Mapping of the QTL for GLS Tolerance

The integrated genetic and physical map of maize was used to identifyall BAC contigs located in the region. Low-copy BAC ends sequences andPHM markers from these contigs were used to develop CAPS markers. It wasdetermined that bacm2.pk027.h10.f and bacm.pk098.d7 flank the GLS locuson one side, and bacm.pk022.b8 and PHM 7245 flank on the other side.

Example 3 Development of PHJEP

A progeny individual derived from the BC4 generation that had the QTLregion, a phenotype most like PHN46, and a good resistance phenotype wasselected. The individual was selfed to ensure a largely homozygousgenome. The resulting inbred was designated PHJEP. The introgression ofthe GLS QTL resulted in a disease resistance score for PHJEP of 4 (on ascale of 1-9) compared to 1 or 2 for PHN46. FIG. 6 demonstrates thephenotypic difference between PHN46 and PH14T following inoculation withCercospora zeae-maydis.

The QTL was further backcrossed into PHN46. A progeny individual derivedfrom the BC7 (dose 8) generation was selected that had the newly definedQTL region and a smaller segment of donor in the region immediatelyflanking the QTL. The individual was selfed to ensure a largelyhomozygous genome. The resulting inbreds were coded PHW6N and PHW6M.These inbreds were found to have GLS tolerance levels equivalent toPH14T and PHJEP. Since PHJEP was developed first and did not exhibitlinkage drag or other deleterious effects due to the QTL introgression,PHJEP was selected as the primary donor for the GLS tolerance QTL overPHW6N and PHW6M.

Example 4 Unique Haplotype in PHJEP

The tropical donor region in PH14T has SNP nucleotides A and A at PHM00045-01 and PHM 00043-01, respectively. The recurrent parent PHN46 hasnucleotides G and G at these same marker loci. Introgression of the GLSQTL from PH14T into PHN46 and subsequent selection for the QTL overmultiple BC generations, while selecting for recurrent parent in theflanking region, resulted in a recombination just above the GLS QTL andthe nucleotides in PHJEP being G (PHN46 allele) and A (PH14T allele) forPHM 00045-01 and PHM 00043-01, respectively. The GLS QTL is located nearthe centromere of chromosome 4 in a region with low recombinationfrequency. The recombination just above the GLS locus in PHJEP isconsidered to be a rare recombination event. In subsequent breedingpopulations between PHJEP and other inbreds, this nucleotide pattern, orhaplotype, has been maintained. During multiple introgressions of PHJEPGLS donor region into elite materials, this recombination has beenmaintained. One haplotype that defines the rare recombination is: PHM00045-01: G; PHM 00043-01: A; PHM 15534-13: A; PHM 04694-10: G; PHM01811-32: T; PHM 01963-15: T; PHM 01963-22: C; PHM 05013-12: T; PHM00586-10: T; and PHM 00049-01: A (see Table 5). A survey of public andproprietary inbreds determined that this haplotype is unique to PHJEP.This allelic combination is unique in Applicants' germplasm.

TABLE 5 Haplotypes for Inbreds PH14T, PHN46, and PHJEP in Chromosome 4Region Containing the GLS QTL PH14T PHN46 PHJEP PHM 00045-01 1.1 3.3 3.3PHM 00043-01 1.1 3.3 1.1 PHM 15534-13 1.1 4.4 1.1 PHM 04694-10 3.3 4.43.3 PHM 01811-32 4.4 2.2 4.4 PHM 01963-15 4.4 2.2 4.4 PHM 01963-22 2.22.2 2.2 PHM 05013-12 4.4 4.4 4.4 PHM 00586-10 4.4 2.2 4.4 PHM 00049-011.1 4.4 1.1 1 = A, 2 = C, 3 = G, 4 = T

Example 5 GLS Analysis of PH14T, PHN46, and PHJEP

PH14T, PHN46, and PHJEP were grown at 4 locations with 8 reps perlocation. Maize plants were evaluated for GLS on a 1 to 9 scale, withone being “poor” and 9 being “good”.

The mean GLS scores, based on a scale of 1 to 9, for PH14T, PHN46, andPHJEP were 5.4, 3.6, and 5.3, respectively. Inbred PHJEP had a 1.5-foldincrease in disease resistance (on a scale of 1-9) as compared to inbredPHN46, and this difference was significant at a p-value≦0.05. InbredPHJEP and the GLS tolerant line PH14T had similar scores for GLS.

Example 6 Conversions of Other Elite Inbreds with PHJEP

PHJEP was used as a donor to convert over 40 different inbreds to haveGLS resistance. Table 6 shows a comparison between three of thesehybrids and three hybrids produced from non-PHJEP sources.

TABLE 6 Exp Hybrid Yield (bu/a) Stagrn GLFSPT PHP38/PHJEP 188.5 193 6PH705/PHJEP 185.8 147 6 PH05F/PHJEP 178.1 166 7 3394 155.9 79 3(PHP38/PHN46) PH705/PHN46 155.7 70 5 PH05F/PHN46 153.3 70 5 CV% 5.9 25.513.6 SED between 2 7 37 1 entry means Advantage 29 ± 4.0 96 ± 19 2.0 ±0.5Yield (bu/a) is yield (bushels/acre), that is, yield of the grain atharvest by weight or volume (bushels) per unit area (acre) adjusted to15% moisture. Stagrn is stay green, that is, the measure of plant healthnear the time of black layer formation (physiological maturity). A highscore indicates better late-season plant health. GLFSPT is gray leafspot, that is, a 1-9 visual rating indicating the resistance to grayleaf spot. A higher score indicates a higher resistance. CV % is thecoefficient of variance (expressed as a percentage). SED is standarderror of the difference.

Example 7 Use of the QTL to Select GLS Tolerant Plants

The SSR markers PHI026 [PHI079 or GPC1 or GAPC1 or NC005], LGI145183[BNLG1937], LGI455751 [BNLG1265], LGI135263 [BNLG1755], EST337321[UMC1175], LGI123864 [BNLG1189] and UMC1346 were used to select for thedonor QTL region from PH14T during successive backcrossing. Phenotypingwas also carried out in BC3 (270 individuals) and BC4 (110 individuals)generations so that the QTL could be further validated and the locationfurther refined.

In the BC4 population the QTL was localized to a region defined byBNLG1755 and UMC1346 (FIG. 2); a distance of 4.4 cM on the IBM2 2004neighbors map. The GLS QTL was found to explain 55.1% of the totalphenotypic variation. The average phenotypic effect of the QTL wasgreater than 2 on the GLS disease scale of 1-9.

Example 8 Efficacy in Hybrids

PHJEP, PHW6N, PHW6M, and the near-isogenic inbred PHN46 lacking the GLSQTL were test crossed to PHP38, PH705, and PH05F. The resulting hybridswere compared in 16 yield test locations, with 3-4 replications perlocation. The locations focused on the eastern corn belt regions.Fourteen locations provided yield data and 13 locations provided GLStolerance data. The GLS tolerant versions of the hybrids were found tohave a consistent yield advantage and increased disease tolerancecompared to the non-tolerant versions (Table 7). In Table 7, 3394 is thehybrid formed by the cross between inbreds PHP38 and PHN46.

TABLE 7 Exp Hybrid Exp Hybrid Yield GLFSPT Check Hybrid Exp HybridAdvantage (bu/a) Reps Advantage Reps 3394 PHP38/PHJEP 20.9 47 2.7 503394 PHP38/PHW6N 9.4 15 2.4 19 3394 PHP38/PHW6M 19.3 4 2.8 12PH705/PHN46 PH705/PHJEP 30.3 6 1.7 7 PH705/PHN46 PH705/PHW6M 13.4 6 1.67 PH705/PHN46 PH705/PHW6N 21.3 6 1.3 7 PH05F/PHN46 PH05F/PHJEP 43.5 62.0 7 PH05F/PHN46 PH05F/PHW6N 40.9 5 1.7 9 PH05F/PHN46 PH05F/PHW6M 36.94 1.4 9For example, hybrids with PHJEP had between 20.9 and 43.5 bu/acre yieldadvantage and 1.7-2.7 increase in disease resistance (on a scale of1-9). FIG. 7 demonstrates the effect of the GLS QTL on hybridresistance.

Example 9 Map Based Analysis of the GLS Tolerance QTL

The markers BNLG1755 and UMC1346 were used to identify the B73 contigs4022, 4023 and 4024 underlying the QTL region. Three CAPs (CleavedAmplified Polymorphism) markers were designed to BACs bacm.pk040.o17(marker PHM 00045), bcb.pk0333.o19 (PHM 00043) and bacm.pk022.b8 (PHM00049) by designing primers to the BAC end sequences and cleaving theresulting amplicons (see FIGS. 3A and 3B). The same BAC end sequenceswere used to design Invader SNP marker assays for application inhigh-throughput molecular breeding. A BC7 population of 4,464 RIindividuals was generated, and the CAPs markers were used to identifyprogeny individuals with recombinants in the region. These progeny werephenotyped for GLS resistance in the field and also selfed to generatefurther progeny and enable more accurate phenotyping. The combinedmarker and phenotypic data were used to localize the QTL to contig 4024.Additional markers were developed to BACs (bacm2.pk065.b22.f,bacc.pk0267.m12.f, bacb.pk0241.h17.f, chp2.pk0007.d2, bacc.pk0530.f13.f,bacb.pk0269.n19, bacb.pk0009.b21.f, bacb.pk0117.i09.f, bacc.pk0280.n12,bacb.pk0219.j20, bacc.pk0132.b16.f, bacb.pk0221.o22, bacb.pk0544.j18 andbacb.pk0540.c18.f) and an EST overgo probe (cl33021_(—)1) within thiscontig. These additional CAPs markers were used to aid in fine mappingand map based cloning.

During the process of fine mapping, a project was initiated to sequenceESTs and BACs within the QTL region using the high throughput 454sequencing technology from Life Sciences. Twenty five overlapping B73BAC clones covering the interval between bcb.pk0333.o19 andp0094.csstg88 were pooled and sequenced by 454 Life Sciences. Shortsequence reads meant that the sequences could not be assembled to createa tiling path, but deep sequence sampling of the BAC clones (average 20×coverage) suggested that the region was fully sequenced. Sequenceannotation failed to identify a sequence resembling a disease resistancegene in this interval.

During this period, additional CAPs markers within the region were usedto further locate the QTL to a region between the EST cl33021_(—)1 andthe BAC end bacb.pk0009.b21.f. This is illustrated in Table 8 where thenumber of recombinants column describes the number of plants out of4,464 progeny individuals that had a recombination between thecorresponding marker and the QTL. Zero recombinants show that the markeris very close to the gene causing the phenotype such that recombinationcannot break the tight linkage between the phenotype (and therefore thegene) and the marker. Zero recombinants out of 4,464 progeny representsa recombination distance of less than 0.02 cM.

TABLE 8 Fine mapping of GLS QTL with 4,464 progeny individuals andmarkers developed to BAC-end sequences Number of BAC Marker Recombinantsbacb.pk0333.o19 PHM 29 00043 bacc.pk0267.m12.f 4 cl33021_1 7bacb.pk0241.h17.f 0 chp2.pk0007.d2 0 p0094.csstg88 0 bacb.pk0269.n19 0Putative R gene bacb.pk0009.b21.f 20 bacb.pk0117.i09.f 20bacc.pk0280.n12 23 bacb.pk0219.j20 25 bacc.pk0132.b16.f 27bacb.pk0221.o22 27 bacb.pk0544.j18 35 bacb.pk0540.c18.f 35 bacm.pk022.b8PHI 00049 39

Two additional BAC clones; bacb.pk0269.n19 and bacb.pk0009.b21.f, weresequenced using the double-stranded random shotgun approach (Bodenteichet al., In “Automated DNA sequencing and analysis techniques” (ed. Adamset al.), pp. 42-50, Academic Press, London, UK (1994)). Briefly, aftereach BAC was isolated via a double-acetate cleared lysate protocol, theclones were sheared by nebulization, and the resulting fragments wereend-repaired and subcloned into pBluescript II SK(+). Aftertransformation into DH-10B electro-competent Escherichia coli cells(InVitrogen) via electroporation, the colonies were picked with anautomatic Q-Bot colony picker (Genetix) and stored at −80° C. infreezing media containing 6% glycerol and 100 μg/ml Ampicillin.

For sequencing, clones were recovered from archived glycerol culturesgrown/frozen in 384-well freezing media plates and replicated with asterile 384 pin replicator (Genetix) in 384-well microtiter platescontaining LB+100 μg/ml Ampicillin (replicated plates). Plasmids werethen isolated, using the Templiphi DNA sequencing template amplificationkit method (Amersham Biosciences). Briefly, the Templiphi method usesbacteriophage φ29 DNA polymerase to amplify circular single-stranded ordouble-stranded DNA by isothermal rolling circle amplification (Dean etal., Genome Res. 11:1095-99 (2001); Nelson et al., Biotechniques32:S44-S47 (2002); Reagin et al., J. Biomol. Tech. 14:143-148 (2003)).Cells were added to 5 μl of dilution buffer and partially lysed at 95°C. for 3 min to release the denatured template. 5 μl of Templiphi premixwere then added to each sample, and the resulting reaction mixture wasincubated at 30° C. for 16 hours, then at 65° C. for 10 min toinactivate the φ29 DNA polymerase activity. DNA quantification with thePicoGreen® dsDNA Quantitation Reagent (Molecular Probes) was performedafter diluting the amplified samples 1:3 in distilled water. Theamplified products were then denatured at 95° C. for 10 min andend-sequenced in 384-well plates, using vector-primed M13oligonucleotides and the ABI BigDye version 3.1 Prism sequencing kit.After ethanol-based cleanup, cycle sequencing reaction products wereresolved and detected on Perkin-Elmer ABI 3730xl automated sequencers,and individual sequences were assembled with the public domainPhred/Phrap/Consed package. While Phred reads DNA sequencing tracefiles, calls bases, assigns a quality value to each called base, andwrites the base calls and quality values to output files, Phrap usesPhred-based sequencing files for assembling shotgun DNA sequence data(see the Laboratory of Phil Green website, Genome Sciences Department,University of Washington). Consed is a tool for viewing, editing, andfinishing sequence assemblies generated with Phred and Phrap (Gordon etal., Genome Res. 8:195-202 (1998)). Contig order was viewed andconfirmed with Exgap (A. Hua, University of Oklahoma, personalcommunication), a local graphic tool that uses pair read information toorder contigs generated by Phred, Phrap, and Consed, and confirms theaccuracy of the Phrap-based assembly.

The assembled sequence contained an annotated protein-coding generesembling a putative R gene of the type classified as an LRR-likeprotein kinase. The annotated sequence is presented in SEQ ID NO:49(with the translated amino acid sequence shown in SEQ ID NO:50). TheCAPs marker for bacb.pk0269.n19 was found to preside in the 3′ end ofthe putative R gene.

What is claimed:
 1. A method of identifying and selecting a maize plantthat shows tolerance to gray leaf spot, said method comprising: a.detecting in a maize plant at a locus linked to any of the followingmarkers: PHM 15534-13, PHM 04694-10, PHM 01811-32, PHM 01963-15, PHM01963-22, PHM 05013-12, and PHM 00586-10, an allele associated with oneor more of the following: i. an A at PHM 15534-13; ii. a G at PHM04694-10; iii. a T at PHM 01811-32; iv. a T at PHM 01963-15; v. a C atPHM 01963-22; vi. a T at PHM 05013-12; and vii. a T at PHM 00586-10; b.selecting a maize plant that has the allele associated with one or moreof i-vii; and c. crossing the maize plant to a second maize plant totransmit the allele associated with one or more of i-vii to at least oneprogeny.
 2. The method of claim 1, wherein the locus is linked to any ofPHM 15534-13, PHM 04694-10, PHM 01811-32, PHM 01963-15, PHM01963-22, PHM05013-12, and PHM 00586-10 by 25 cM on an IBM2 map.
 3. The method ofclaim 1, wherein the locus is linked to any of PHM 15534-13, PHM04694-10, PHM 01811-32, PHM 01963-15, PHM01963-22, PHM 05013-12, and PHM00586-10 by 10 cM on an IBM2 map.
 4. The method of claim 1, wherein thelocus is linked to any of PHM 15534-13, PHM 04694-10, PHM 01811-32, PHM01963-15, PHM 01963-22, PHM 05013-12, and PHM 00586-10 by 5 cM on anIBM2 map.
 5. A method of identifying and selecting a maize plant thatshows tolerance to gray leaf spot, said method comprising: a. detectingin a maize plant one or more of the following: i. an A at PHM 15534-13;ii. a G at PHM 04694-10; iii. a Tat PHM 01811-32; iv. a Tat PHM01963-15; v. a C at PHM 01963-22; vi. a Tat PHM 05013-12; and vii. a TatPHM 00586-10; and b. selecting a maize plant that has one or more ofi-vii; and c. crossing the maize plant to a second maize plant totransmit one or more of i-vii to at least one progeny.
 6. A method ofmaking a progeny maize plant with tolerance to gray leaf spot, themethod comprising: a. obtaining a first parent maize plant thatcomprises within its genome one or more marker alleles selected from thegroup consisting of: i. an A at PHM 15534-13; ii. a G at PHM 04694-10;iii. a Tat PHM 01811-32; iv. a Tat PHM 01963-15; v. a C at PHM 01963-22;vi. a Tat PHM 05013-12; and vii. a Tat PHM 00586-10; b. crossing saidfirst parent maize plant to a second parent maize plant; c. growing theparent maize plant used as a female under plant growth conditions toyield maize plant progeny; d. assaying the maize plant progeny for thepresence of any of the marker alleles set forth in i-vii in step (a) orthe presence of an allele linked to any of the marker alleles set forthin i-vii in step (a), wherein the marker alleles set forth in i-vii ofstep (a) or an allele linked to any of the marker alleles set forth ini-vii of step (a) are associated with tolerance to gray leaf spot; ande. selecting progeny plants that possess any of the marker alleles setforth in i-vii in step (a) or an allele linked to any of the markeralleles set forth in i-vii in step (a).